作者想感谢查尔斯Luetje博士提供的T -迷宫和行为房间。这项工作是由美国心脏协会（遐），国立神经系统疾病和中风格兰特R01NS64269（RGZ），和皮尤慈善信托基金（RGZ）的支持。

1。简介在这段视频中，我们将展示两个行为实验用果蝇表现出测量运动和学习记忆功能，在神经退行性疾病的损害。首先，我们将呈现怎样的运动行为，可以使用负geotaxis检测。其次，我们将评估使用正趋光，进行培训，以联营公司与厌恶苦味的光，然后进行测试，6小时后，以评估记忆功能的果蝇的学习和记忆能力。最后，我们将讨论这些行为检测的影响和限制。 2。设备和试剂<ol><li>飞行工作<ul><li>立体声范围（蔡司） </li><li>推动了苍蝇的小画笔</li><li> 果蝇的二氧化碳（CO 2）麻醉器具（Geneseesci公司） </li></ul></li><li>负Geotaxis含量<ul><li> 28.5 × 95毫米的聚苯乙烯瓶（国会瓶，公司） </li><li>迷你报警定时器/秒表（VWR国际） </li><li> Sharpie笔</li><li>胶带</li></ul></li><li>厌恶的趋光性抑制法<ul><li>奎宁盐酸盐（Sigma公司Aldrich公司; CAS编号：6119-47-7） </li><li>蒸馏水</li><li>按重量计算精确的稀释数字测量规模</li><li>快速断开连接器，高密度聚乙烯（NALGENE VWR目录号62868-021） </li><li> 15 mL离心管中（VWR International的），从封闭式的2毫升大关，快速断开连接适配器适合光源</li><li>单通道200微升pipetter加载到过滤纸衬腔奎宁解决方案</li><li> T -迷宫（简单的行为系统，图1） </li><li>铝箔</li><li>光源（蔡司） </li><li>与红色光灯。 </li><li>滤纸</li><li>迷你报警定时器/秒表（VWR国际） </li></ul></li></ol> 3。准备苍蝇<ul><li>苍蝇标准玉米面琼脂糖蜜酵母中保持在25 ° C下12小时的光/暗周期。 </li><li>处女蝇羽化当天的碳二氧化碳麻醉下被隔离。老化研究，果蝇维持在每小瓶20组，并转移到一个新的小瓶，每3天。重要的是使用来自同一父母老化研究的交叉的兄弟姐妹苍蝇，以减少遗传背景所产生的分歧。 </li><li>对于这两种实验中，苍蝇是绝育手术和维持在10％小瓶苍蝇。行为上的性别差异是显着的，最好是不要混用雄性和雌性果蝇在一项研究中，作为数据可能很难解释和重现6。 </li><li>负geotaxis检测，苍蝇到每小瓶10个组进行排序和测试麻醉后一小时。重要的是苍蝇从麻醉中完全恢复，因为这可能影响的运动活动。 </li><li>为APS检测，各组的果蝇被放置在一个空聚苯乙烯小瓶水滤纸沾了六个小时前进行检测。这确保了苍蝇饿死之前检测和厌恶的味道会更加敏锐的。只是前进行检测，是摆在每个飞从一组十一个空的15ml离心管，然后封顶松散，以避免逃生。 </li></ul> 4。负Geotaxis含量<ul><li> Geotaxis一般测十到二十组十个人同一基因型或治疗（100-200苍蝇每个基因型/治疗总）7。十大女性或男性苍蝇的CO 2麻醉设备和放置在一个单独的小瓶各组的排序群体。允许苍蝇从麻醉中恢复的至少一个小时。 </li><li>准备为每个​​组的登山设备，这样两个空的聚苯乙烯瓶垂直面对对方磁带加入。重要的是要确保，小瓶的开口是完全相互对齐，提供一个苍蝇，甚至攀岩表面。 </li><li>对于较低的小瓶，衡量一个8厘米以上的底部表面的垂直距离和标记每一瓶小瓶整个圆周周围绘制一个圆。 </li><li>转移到下仔细地防止任何飞逃逸小瓶一组十个苍蝇。立即盖样品瓶和磁带，牢固地附近的联系开口较低的小瓶。允许苍蝇前进行检测，使其恢复到新的设置为1分钟。 </li><li>轻轻拍打苍蝇瓶底部和措施的苍蝇，可以爬10秒8厘米以上的自来水后。 </li><li>重复这个实验中，对同一组十倍允许为1分钟的休息时间，在每个审判。 </li><li>记录每个组的苍蝇数量，通过为8厘米的标志总苍蝇的百分比。 </li></ul> 5。厌恶的趋光性抑制含量（APS含量） 来自乐布雷斯和Buecher（2002）4改编，本实验利用果蝇正趋光行为，培养他们联想到与厌恶刺激（在这种情况下，苦味的奎宁） 。野生型果蝇的培训/调节阶段后，将能联想到点燃的面积和厌恶的味道，避免它。学习能力受损的果蝇会失败，使这个协会。此外，APS的检测也可以被用来衡量短期记忆功能的苍蝇已经受到训练有素的苍蝇以相同的测试后6小时空调，测试自己的能力，记住所学的任务5。 <ol><li>准备奎宁解决方案<ul><li>奎宁盐酸溶解在蒸馏水中，使一个0.1M原液（1.98克于50ml蒸馏水）。在-20 ° C原液可保持长达一年的小等份</li><li>准备一个1微米的工作解决方案，在蒸馏水稀释的原液。 </li></ul></li><li>准备T -迷宫<ul><li> T -迷宫陷阱门和两个独立的商会，一个“黑暗”室，“点燃”室（图1）由中心柱。 </li><li>商会准备：取​​两个15毫升塑料离心管中，并切成2毫升标记从底部封闭与手册年底看到。适合的两端连接适配器，并用封口膜密封连接。管端的适配器作为输入插槽鹅颈光源。与光源连接管和管将“点燃”室。包装用铝箔作为“黑马”商会的其他管。 </li><li>加入180uL无论是蒸馏水或奎宁溶液过滤器在光室纸张和地点。 </li><li>旋中心柱两侧的照明和黑暗商会组装T -迷宫，在中间封闭（图1）陷阱门。 </li><li>插入点燃室的光源。 </li></ul></li><li>训练苍蝇的趋光性抑制与厌恶的刺激<ul><li>拧下从T -迷宫暗腔，并转移到这个会议厅内飞，并立即拧暗管的迷宫。 </li><li>关闭在房间里的灯和转向红灯。 </li><li>允许飞往逐渐适应在30秒的暗腔，慢慢打开照亮源点燃室。慢慢打开活门分隔两院。 </li><li>如果飞各界在10秒内点燃室，它被认为是积极的趋光，准备检测培训。在趋光性的失败表明在视觉系统的问题和苍蝇与负趋光性，需要从检测中排除。 </li><li>厌恶的趋光性抑制（APS）培训，挖掘飞，表现出积极的趋光性暗腔，关闭活门和关灯。允许30秒的驯化。在这段时间内点燃室与奎宁溶液的滤纸。慢慢打开活门，并把灯打开。允许飞步行到奎宁涂点燃室。一分钟后，点选飞暗腔，并重复这9次以上。 （通常情况下，野生型果蝇避免点燃后3至5个培训试验室）。 </li><li>训练结束后，立即将进行五个测试试验。在每个测试试验中，让训练有素的飞行，步行到点燃室，10秒后，指示灯会亮起。如果步行到点燃的小瓶，将被记录为“通行证”，这是相当于“通过加固任务教训”。合格率连续超过五年的审判记录，如下面图2为PC0（0小时后空调）所示。 </li></ul></li><li>评估在果蝇的短时记忆功能<ul><li>每个飞行训练和初步的PC0录音后，放回其原来的食品瓶，并保持预留了六个小时。 </li><li>六个小时的培训后，受每个飞5试验再像以前一样的方式，并记录的次数飞避免（通过）或进入（失败）点燃的小瓶。这个合格率记录为PC6（6小时后空调），这是短期记忆的一个指标。 </li></ul></li></ol> 6。数据的解释和统计分析<ol><li>负Geotaxis含量<ul><li>从这个实验中产生的原始数据，穿越8厘米的标志，在10秒后，各组的苍蝇数量。转换成百分比，并计算各组的平均合格率超过10节。 </li><li>每组的平均合格率与平均值的标准误差（SEM）（基因型，治疗等）的数据用图形表示。 </li><li>群体之间的差异然后可以被确定为显着性或不使用学生t检验或方差分析。 </li></ul></li><li>厌恶的趋光性抑制法<ul><li>从这个实验的原始数据表示为每飞行的次数，避免点燃连续5试验室。的合格率的计算公式为总试验成功避免试验的百分比。以这种方式计算最低15％组苍蝇的平均合格率。 </li><li>数据以图形方式表示为平均合格率的PC0（后立即空调;“学习”的指标）或PC6的平均合格率（后6小时空调;“记忆”的指标）与各自的扫描电镜。 </li><li>与野生型和基因操纵良性“组（如绿色荧光蛋白的过度表达苍蝇）适当团体之间的学习和记忆指数比较。 </li></ul></li></ol> 7。代表性的成果在这个视频实验中，我们正在评估在中枢神经系统的学习记忆和运动障碍，在果蝇中过表达的人类头，并比较他们的表现苍蝇高表达绿色荧光蛋白的。已被证明在果蝇神经元的过度表达人类头，导致大脑中的 8严重空泡，导致学习和记忆功能 9的重大赤字。如图2a所示，10日龄的男性或女性过度表达人类头苍蝇有显着的运动功能的赤字相比，年龄相匹配的绿色荧光蛋白过度表达的苍蝇。此外，这些果蝇没有关联苦味PC0合格率对照组相比，作为以及不能记住这个协会由PC6的合格率（图2B）下降所示。本实验进一步证实， 果蝇模型可以用来研究人类神经退行性条件，并扼要一些在人类和哺乳动物中观察到的行为表型。 <img src="/files/ftp_upload/2504/2504fig1.jpg" alt="图1" /> 图1。 T -迷宫设置厌恶的趋光性抑制法。整体APSA连接“点燃”猎鹰管，衬有滤纸和左侧（在箔覆盖）的“暗”管，一个活门分隔光源实验的实验装置。 B.在训练和测试阶段，飞在黑暗中商会和陷阱门开了，在点燃室后，指示灯会亮起。 <img src="/files/ftp_upload/2504/2504fig2.jpg" alt="图2" /> 图2。代表从反面geotaxis和使用APS实验结果苍蝇与泛神经驱动器的高表达绿色荧光蛋白或的头。负geotaxis检测，10天岁的男性（蓝色列）或女性（红色柱）苍蝇高表达绿色荧光蛋白表现出较高的登山活动，比头高表达苍蝇。 B. 20日龄雌性果蝇高表达绿色荧光蛋白表现出更好的学习（PC0）和内存（PC6）比头高表达苍蝇的功能。

<ol>
	<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> Annual Review of Neuroscience. 26, 627-656 (2003).</li><li>Lloyd, T. E. &. a. m. p. ;. a. m. p., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li><li>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=Behavioral+mutants+of+Drosophila+isolated+by+countercurrent+distribution.">Behavioral mutants of Drosophila isolated by countercurrent distribution.</a> Proc. Natl. Acad. Sci. USA. 58, 1112-1119 (1967).</li><li>Le Bourg, E., Buecher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learned+suppression+of+photopositive+tendencies+in+Drosophila+melanogaster.">Learned suppression of photopositive tendencies in Drosophila melanogaster.</a> Animal Learning &#38; Behavior. 30, 330-341 (2002).</li><li>Seugnet, L., Suzuki, Y., Stidd, R., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aversive+phototaxic+suppression:+evaluation+of+a+short-term+memory+assay+in+Drosophila+melanogaster.">Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila melanogaster.</a> Genes Brain Behav. 8, 377-389 (2009).</li><li>Nilsen, S. P., Chan, Y. -. B., Huber, R., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender-selective+patterns+of+aggressive+behavior+in+Drosophila+melanogaster.">Gender-selective patterns of aggressive behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 101, 12342-12347 (2004).</li><li>Leal, S. M., Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evidence+for+GABAergic+regulation+of+specific+behaviors+in+Drosophila+melanogaster.">Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster.</a> J Neurobiol. 50, 245-261 (2002).</li><li>Wittmann, C. W., Wszolek, M. F., Shulman, J. M., Salvaterra, P. M., Lewis, J., Hutton, M., 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=Tauopathy+in+Drosophila:+Neurodegeneration+Without+Neurofibrillary+Tangles.">Tauopathy in Drosophila: Neurodegeneration Without Neurofibrillary Tangles.</a> Science. 293, 711-714 (2001).</li><li>Mershin, A., Pavlopoulos, E., Fitch, O., Braden, B. C., Nanopoulos, D. V., 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=Learning+and+memory+deficits+upon+TAU+accumulation+in+Drosophila+mushroom+body+neurons.">Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons.</a> Learn Mem. 11, 277-287 (2004).</li><li>Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+Suppression+Involving+Behavioral+Mutants+with+Altered+Nerve+Excitability+in+DROSOPHILA+MELANOGASTER.">Indirect Suppression Involving Behavioral Mutants with Altered Nerve Excitability in DROSOPHILA MELANOGASTER.</a> Genetics. 100, 597-614 (1982).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholinesterase+mutants+in+Drosophila+and+their+effects+on+the+structure+and+function+of+the+central+nervous+system.">Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system.</a> J Comp Neurol. 189, 741-774 (1980).</li></ol>

既是消极geotaxis和APS检测强有力的行为分析来衡量运动器官或遗传或环境操纵产生的学习和记忆能力的变化。不过，也有一些有待进一步优化这些实验的缺点。 在负geotaxis检测，某些基因的突变可以使果蝇严重的运动功能障碍。苍蝇在这种情况下，难以攀登的墙壁，在搅拌后的重力。直接测量运动的其他行为分析，可能更为合适。这些实验包括翻正反射行为的措施瘫痪，并检获10和11 optomotor行为记录失去运动协调。 在APS法，第一个障碍，可随机点燃室门口停不前进的苍蝇。这将成为一个问题，如果老龄化研究的条件之一，因为旧的苍蝇一般较不活跃，往往会停下来休息很长一段时间。在这种情况下，实验者提供机械性刺激，以促进运动，这并不总是有效的的。其次，可能有人会说，去点燃室从积极的趋光性的损失结果失败。这是很少见的情况下，由于以前的工作表明，苍蝇回头光响应一旦负强化是删除4。第三，接触厌恶刺激的持续时间可能会有所不同从审判审判。这种变化通常可以减少浸泡滤纸包围室的内壁，以防止苍蝇避免奎宁。此外，实验者可以确保每个飞行花费足够的时间增加金额另有说明1分钟的时间花费在会议厅内，用滤纸接触。 总之，负面geotaxis和APS检测评估行为赤字的快速和简单的方法和定量表示运动功能和果蝇的学习和记忆能力。这些检测建模在果蝇的人类神经退行性条件，特别是在有用的。

<table border="1"><tbody><tr><th>材料</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td>聚苯乙烯瓶</td><td>国会瓶，公司</td><td> 28Fly </td><td> 28.5 × 95毫米聚苯乙烯瓶;材料的质量很重要，坏的，如果太滑苍蝇爬。 </td></tr><tr><td>解剖立体</td><td>蔡司</td><td> 000000-1432-979 </td><td>鹅颈2分支光导（12V/30W） </td></tr><tr><td>奎宁盐酸</td><td>西格玛爱秩序</td><td> 6119-47-7 </td><td>溶解于蒸馏水。 </td></tr><tr><td>快速断开连接器</td><td> NALGENE（VWR） </td><td> 62868-021 </td><td>高密度聚乙烯</td></tr><tr><td> T -迷宫</td><td>简单的行为系统</td><td></td><td></td></tr><tr><td>单通道200微升吸管</td><td> Eppendorf公司</td><td> 022443305 </td><td></td></tr></tbody></table>

assaying locomotor, learning, and memory deficits in drosophila models of neurodegeneration

在遗传方式的进步使在涉及人类神经退行性疾病，利用果蝇作为模型系统1基因的研究。大多数这些疾病，包括老年痴呆症，帕金森氏症和亨廷顿氏病的特点是年龄依赖性，在学习和记忆功能和运动协调 2恶化。在这里，我们使用行为的分析 ，包括3负geotaxis检测和厌恶phototaxic抑制的测定（APS法）4,5，表明一些与人类神经退行性疾病相关的行为特征，可以在果蝇扼要。在负geotaxis检测，苍蝇的自然倾向对重力时，激动是用来研究基因或条件，可能会妨碍的运动能力。在APS分析，学习和记忆功能的测试正趋光飞行训练关联厌恶苦味轻，从而避免这种情况，否则自然的倾向，走向光明。培训后6个小时是用来评估记忆功能测试这些受过训练的苍蝇。使用这些检测，对发展神经退行性疾病的任何遗传或环境因素的贡献可以很容易地在果蝇研究。

Watch this Scientific Journal Video about Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration at JoVE.com

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

行为分析测量的运动功能，学习和记忆能力<em&gt;果蝇</em&gt;。

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 ...

assaying-locomotor-learning-memory-deficits-drosophila-models

Research

JoVE Journal

Neuroscience

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

33.2K 次观看.  University of Miami, Miller School of Medicine. 行为分析测量的运动功能，学习和记忆能力果蝇。

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

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

科研

神经科学

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

在阿尔茨海默氏病模型小鼠学习和记忆障碍的Morris水迷宫测试

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

果蝇嗅觉成人学习的冲击

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

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 ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

评估空间学习记忆小鳞类爬行动物

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

含量的运动能力，学习和记忆的赤字果蝇模型