我们要特别感谢他们的实验装置和意见的手稿上的技术指导，的格柏实验室的成员。我们也感谢柳博芙·Pankevych飞的维修保养及野生型各州股票。这项工作是支持的DFG授予，的SNF授予31003A_132812 / 1和康斯坦茨大学的Zukunftskolleg（TH1584/1-1所有的AST）。

1。准备<ol><li> 果蝇野生型幼虫被提出，在25°C和60％-80％的湿度在14/10光/暗周期。对于控制的确切年龄的幼虫始终成一小瓶（6厘米的高度和直径2.5 cm），其中包括约6毫升的标飞的食物，男10例，女20例。苍蝇产卵12小时，并在第二天被转移到一个新的小瓶。 5-6天后产蛋幼虫达到馈送第三龄期，如果在25℃引发，现在可以被用于行为实验。然而，一个具有确保只幼虫，仍然是在食品和不是从侧面的小瓶的幼虫。这些幼虫已经达到了"流浪3龄阶段" -在短期内化蛹前-和它们的用法的解释复杂化的结果。 </li><li>制备的2.5％琼脂的培养皿中（其他实验室用1％的琼脂浓度在整个实验过程中，如何越来越低的浓度可能允许投喂3 龄幼虫挖底物）:称取2.5 g琼脂糖凝胶在100毫升DDH 2 O烧热在微波炉中的溶液，直到它开始沸腾。小心地搅拌溶液，将它放入微波炉加热，直到所有的琼脂糖溶解。倒入热的琼脂糖溶液倒入培养皿的陪替氏培养皿的底部，使得被完全覆盖的琼脂糖溶液形成光滑的表面。让溶液冷却至室温，并关闭盖子。不要马上关闭盖子，因为这将允许冷凝水的盖子。 </li><li> 2M果糖培养皿的制备:称取2.5 g琼脂糖在100毫升DDH 2 O（再次，使用1％的琼脂浓度是可能的，但它可能会允许饲养3龄幼虫挖成的底物）。烧热在微波炉中的溶液，直到它开始沸腾。仔细搅拌解决方案，并把它放回微波，直到所有的琼脂糖溶解。小心热溶液中加入35克的果糖，，慢慢搅拌混合物，直到糖溶解，避免沸腾迟缓。倒入热的果糖琼脂糖溶液倒入陪替氏培养皿的底部，使得被完全覆盖和果糖的琼脂糖溶液形成光滑的表面。让溶液冷却至室温，并关闭盖子。不要马上关闭盖子，因为这将允许冷凝水的盖子。 </li><li>制备的1 - 辛醇（OCT）的气味容器:填写10微升纯十月到一个定制特氟隆气味容器和关闭它的盖，有几个小的孔，以允许蒸发的气味。容器的详细描述给定的格柏和施托克尔2007中。准备三个气味容器10月。气味容器允许插入的化学品的蒸发，但避免幼虫可以直接联系他们。因此，在这里描述的实验的具体解决嗅觉学习，幼虫不会扰动味觉的副作用。 </li><li>戊酯（AM）的气味容器的制备:取AM 1:50石蜡油。填充到一个自定义自制特氟隆气味容器的稀释液的10微升，并关闭它的盖，有几个小的孔，以允许蒸发的气味。准备三个气味的容器AM。的稀释是很重要的现实的原因，即避免强烈的气味比其他可能掩盖了学习相关的变化，两者之间的相对偏好气味的偏好。两个气味的平等的吸引力可能需要将之前的实验中，通过施加与天真动物的稍后描述的测试（2.5）在实验室中证实。这里介绍的值是基于一些出版物的Gerber实验室最近被复制我们的实验室9,15,16。 </li><li>标签的培养皿中的行为学实验之前，所有的培养皿中进行编码。这意味着，果糖含有培养皿有被标记为与一个"X"或一个"A"和琼脂糖只培养皿与​​一个"Y"或"B"的例子。此代码应该被揭露出来后，所有数据都被记录的实验者。通过执行实验"盲"，因此不可能的实验者的预期可能会影响性能的幼虫。为了简化理解为广大的读者，下面我们将谈论的气味作为条件刺激（CS1，CS2），奖励时，果糖培养皿（+）或无报酬时，仅琼脂糖陪替氏培养皿（ - ）。 </li></ol> 2。糖奖励培训和测试<ol><li>收集30投喂3 龄幼虫的食品瓶。转入他们的第一个培养皿中，包含了一些滴自来水，小心地向前和向后移动，用刷子。将它们传输到第二个培养皿中，也包含了一些滴自来水华盛顿，以检查，没有食物糊仍然幼虫的体壁，，否则幼虫将能够在实验过程中体验到食物的气味。这可能会掩盖在学习过程中，他们的表现在测试的情况。 </li><li>培训:培训幼虫相关联的气味，食欲糖提示下制度。沽上的左和右侧的一个"X"标记的OCT气味容器-因此，含果糖奖励的陪替氏培养皿（+）（"盲"实验中，详细信息，请参阅1.6）。组30投喂3 龄幼虫上的培养皿中，盖上盖子，等待5分钟，而动物暴露华侨城。确保幼虫被困在里面的水滴，可以克服表面张力的。 ，幼虫可以自由移动培养皿上，体验嗅觉和/或味觉的刺激。 </li><li>培训:从培养皿中取出幼虫用湿刷和转移米到第二培养皿]是与一个"Y"标记-因此，琼脂糖只（ - ）含有培养皿-和位于它的左侧和右侧的容器具有的AM气味。盖上盖子，等待5​​分钟，而动物暴露于AM。 </li><li>培训:重复2.2）和2.3）的两倍，所有30个幼虫经历了三个训练周期:CS1 /（+） - CS2 /（ - ），（+） - CS1 / CS2 /（ - ）; CS1 /（+） - CS2 /（ - ）。在这个实验中，，CS1表示OCT和CS2代码为AM。 </li><li>测试:将一个AM和一个OCT气味，容器的网站的琼脂糖只的培养皿上的反对。受过训练的动物转移到测试培养皿中间。盖上盖子，等待5​​分钟。随后的幼虫的数目进行计数的左侧，中间和右侧的侧的测试培养皿。 </li><li>重复步骤2.1）2.5），第二组30投喂3 龄幼虫，但交换实验的角色的AM和华侨城等，这些动物会收到以下培训:CS2/（+） - CS1 /（ - ）; CS2 /（+） - CS1 /（ - ）; CS2 /（+） - （ - ）CS1 /。在这个实验中，，CS1表示OCT和CS2代码为AM。 </li><li>可能安排的培训。以上我们提出了一个培训存在的三个培训试验或OCT /（+） - AM /（ - ）/（ - ）或在对等组的三个培训试验的AM /（+） - OCT。然而，为了避免在训练序列依赖性的影响，重要的是不同的序列的以下重复完整的实验中的刺激。通过改变CS1或CS2顺序和奖励文稿在所述第一或第二呈现板，四个不同的序列的训练试验是可能的: </li></ol><table border="1"><tbody><tr><td> 第一组 </td><td> CS1 /（+） - CS2 /（ - ） </td><td> 交换组 </td><td> CS2 /（+） - CS1 /（ - ） </td></tr><tr><td></td><td> CS1 /（ - ） - CS2 /（+） </td><td></td><td> CS2 /（ - ） - CS-1 /（+） </td></tr><tr><td></td><td> CS2 /（+） - CS2 /（ - ） </td><td></td><td> CS1 /（+） - CS2 /（ - ） </td></tr><tr><td></td><td> CS2 /（ - ）（+） - CS1 / </td><td></td><td> CS1 /（ - ） - CS2 /（+） </td></tr></tbody></table>为了防止系统的周边的实验环境刺激的影响，应该执行其中之一的测试中的二分之一的情况下的条件，使得OCT提出，在左边和AM到右侧。在其他的情况下的一半AM应呈现在左侧和OCT在右边。 3。测试任务相关的感官电机学院上述实验的设计允许在野生型馈送3龄幼虫，在其自己的分析臭味糖学习。然而，在日常的实验室生活研究者通常使用两个或更多个不同的实验组的幼虫的比较，如果嗅觉学习取决于看齐在开发过程中添加满足特殊基因，一组特定的神经细胞，突变体的股票，一种特殊的食品的饮食，不同的饲养条件下，有毒化学品，等等。因此，在所有情况下，当两个或多个测试实验组的幼虫做了强制控制实验来测试，如果不同群体的幼虫显示正确的感觉运动视力。这将成为强制性的，并不一定是由于潜在的表型是减少或取消的能力，联营公司与糖的气味。相反，可以根据潜在的学习缺陷的缺陷，在感官电机电路的任何步骤中的处理中的气味和/或糖。或者换句话说，如果一个突变的幼虫是无法感测糖，它不能建立一个糖存储器。但是，这并不让得出的结论是，幼虫不能学习。详细的对照实验，测试OCT，AM和果糖，适当处理的转基因幼虫，有许多工作要做。 1。测试天真OCT preference 收集30投喂3 龄幼虫的食品瓶。仔细地把它们洗干净的自来水2.1。琼脂糖培养皿中的一个侧面上，提出一个单一的的的OCT气味容器的幼虫上的培养皿中，盖上盖子，等待5​​分钟，这样，幼虫可抓取的培养皿东方对OCT气味源。随后幼虫的数目进行计数的左侧，中间和测试的陪替氏培养皿上的右侧。 2。为天真AM偏好测试收集30投喂3 龄幼虫的食品瓶。仔细地把它们洗干净的自来水2.1。琼脂糖培养皿中的一个侧面上，提出一个单一的AM气味容器的幼虫上的培养皿中，盖上盖子，等待5​​分钟，这样，幼虫可抓取的培养皿东方对AM气味源。随后数一数升arvae的左侧，中间和测试的陪替氏培养皿上的右侧。 3。测试幼稚糖偏好收集30投喂3 龄幼虫的食品瓶。仔细地把它们洗干净的自来水2.1。准备陪替氏培养皿在一个一半和2M果糖琼脂糖混合物中的另一半，包含2.5％的琼脂糖。添加到培养皿中的幼虫，盖上盖子，等待5​​分钟，这样，幼虫可抓取的的培养皿东方向的果糖含侧。随后幼虫的数目进行计数的左侧，中间和测试的陪替氏培养皿上的右侧。 制备的一半半培养皿:准备上述1.2节中的普通琼脂糖平板。当琼脂糖填充培养皿被冷却下来，仔细切断琼脂糖用手术刀沿垂直轴。删除一半的琼脂糖从陪替氏培养皿中。一西元一个热果糖的琼脂糖溶液（制备见1.3）的陪替氏培养皿的空半。要特别小心，两个半匹配，并没有形成一个定义的边缘-这会影响幼虫的选择行为，使行为分析相当困难的。4。 深培训尽管测试，如果转基因投喂3 龄幼虫最近一直在野生型水平之间的OCT和空气（3.1），AM和空气（3.2）和糖和纯琼脂糖（3.3），一组额外的测试实验能够区分介绍（讨论Gerber和斯托克，2007年）。这些实验的基本原理如下。在培训期间幼虫进行大量的处理和连续的气味和糖刺激。因此，这是非常可能的，所观察到的学习表型是误导性的（虽然天真气味和糖感知试验上的野生型水平！）。事实上，它是可能的转基因动物ðiffer从野生型幼虫的抗应激能力，动机，疲劳，感觉适应，情境学习和饱腹感的变化。因此，米歇尔斯等人 （2005）提出控制测试是否一个给定的突变体能够:（1）检测AM与一个空气味容器中的幼虫，如果你把在训练中完全一样，除非你省略的奖励，而只是暴露在两个气味;（4（2）检测后，同样的制度，OCT（3）检测与一个空气味容器上午，如果你对幼虫的培训方式，除非你忽略的气味，仅仅暴露的奖励; ）检测到十月后，相同的方案。对于一个全面的讨论和方法的进一步详细信息，请参阅米歇尔斯等人 （2005年）和Gerber和储（2007）。 4。数据分析糖悬赏学习<ol><li>为了评估糖奖励学习协议的数据计算出OCT偏好指数（PREF OCT）的每个的两个往复运动美云的训练组: 第一组接受OCT /（+） - AM /（ - ）培训: PREF华侨城（OCT + / AM）=（的幼虫在OCT侧的 - ＃幼虫AM侧）/所有的幼虫在左，右，中区 对于第二组，AM /（+） - OCT /（ - ）培训: PREF调幅（AM + / OCT）=（＃AM侧 - ＃幼虫幼虫在OCT侧）/＃的所有幼虫在左，右，中区</li></ol><ol start="2"><li>计算性能指数（PI）两个PREF值4.1）。的PI联想学习的气味和惩罚暴露的时间和处理，通过消除扰动的影响: PI =（PREF华侨城（OCT + / AM）+:PREF AM（AM + / OCT））/ 2 因此，督察范围从-1到1之间。显着的负值代表厌恶学习，而显着的正面的价值观描述食欲的学习。一个完整的实验通常包括10个或更多的督察。数据可视化的箱线图includin克的所有值给定的实验组。位于内盒的50％的值被表示为粗线内的箱形图的性能指标中位数。 </li></ol> 5。数据分析任务相关的感官电机学院<ol><li>评估数据进行测试时，适当的的OCT气味处理计算OCT气味的偏好指标如下: 气味PREF华侨城（OCT侧的幼虫 - 幼虫的另一边）= /＃的所有幼虫在左，右，中区</li><li>评估数据进行测试时，进行适当的AM的气味感知计算AM气味的偏好指标如下: 气味PREF AM =（AM侧的幼虫 - 幼虫的另一边）/左，右和中间区域内的所有幼虫</li><li>评估时的数据测试适当的果糖感知的计算的果糖偏好指数如下: PREF果糖=（＃幼虫的另一边的幼虫果糖侧 -）/＃内的左，右和中间区的所有幼虫</li><li>假培训详情米歇尔斯等人，2005年中给出。 </li></ol>

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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+role+of+dopamine+in+Drosophila+larval+classical+olfactory+conditioning.">The role of dopamine in Drosophila larval classical olfactory conditioning.</a> PLoS One. 4, e5897 (2009).</li><li>Neuser, K., Husse, J., Stock, P., Gerber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetitive+olfactory+learning+in+Drosophila+larvae:effects+of+repetition,+reward+strength,+age,+gender,+assay+type+and+memory+span.">Appetitive olfactory learning in Drosophila larvae:effects of repetition, reward strength, age, gender, assay type and memory span.</a> Animal Behaviour. 69, 891-898 (2005).</li><li>Scherer, S., Stocker, R. 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U.S.A. 94, 5131-5136 (1997).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> J. Neurobiol. 47, 81-92 (2001).</li><li>Schroll, C., 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=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr. Biol. 16, 1741-1747 (2006).</li><li>Rosenzweig, M., 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=The+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li><li>Baines, R. A., Uhler, J. P., Thompson, A., Sweeney, S. T., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+electrical+properties+in+Drosophila+neurons+developing+without+synaptic+transmission.">Altered electrical properties in Drosophila neurons developing without synaptic transmission.</a> J. Neurosci. 21, 1523-1531 (2001).</li><li>Chen, Y. C., Mishra, D., Schmitt, L., Schmuker, M., Gerber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+odor+similarity+"space"+in+larval+Drosophila.">A behavioral odor similarity "space" in larval Drosophila.</a> Chem. Senses. 36, 237-249 (2011).</li><li>Saumweber, T., Husse, J., Gerber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+attractiveness+and+associative+learnability+of+odors+can+be+dissociated+in+larval+Drosophila.">Innate attractiveness and associative learnability of odors can be dissociated in larval Drosophila.</a> Chem. Senses. 36, 223-235 (2011).</li><li>von Essen, A. M., Pauls, D., Thum, A. S., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+visual+classical+conditioning+in+Drosophila+larvae.">Capacity of visual classical conditioning in Drosophila larvae.</a> Behav. Neurosci. , (2011).</li><li>Honjo, K., Furukubo-Tokunaga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cAMP+response+element-binding+protein-dependent+medium-term+memory+by+appetitive+gustatory+reinforcement+in+Drosophila+larvae.">Induction of cAMP response element-binding protein-dependent medium-term memory by appetitive gustatory reinforcement in Drosophila larvae.</a> J. Neurosci. 25, 7905-7913 (2005).</li><li>Honjo, K., Furukubo-Tokunaga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+neuronal+networks+and+biochemical+pathways+for+appetitive+and+aversive+memory+in+Drosophila+larvae.">Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae.</a> J. Neurosci. 29, 852-862 (2009).</li><li>Khurana, S., 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=Olfactory+Conditioning+in+the+Third+Instar+Larvae+of+Drosophila+melanogaster+Using+Heat+Shock+Reinforcement.">Olfactory Conditioning in the Third Instar Larvae of Drosophila melanogaster Using Heat Shock Reinforcement.</a> Behav. Genet. 42, 151-161 (2012).</li><li>Tully, T., Cambiazo, V., Kruse, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+through+metamorphosis+in+normal+and+mutant.">Memory through metamorphosis in normal and mutant.</a> 14, 68-74 (1994).</li><li>Michels, B., 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=Cellular+site+and+molecular+mode+of+synapsin+action+in+associative+learning.">Cellular site and molecular mode of synapsin action in associative learning.</a> Learn Mem. 18, 332-344 (2011).</li><li>Saumweber, T., 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=Behavioral+and+synaptic+plasticity+are+impaired+upon+lack+of+the+synaptic+protein+SAP47.">Behavioral and synaptic plasticity are impaired upon lack of the synaptic protein SAP47.</a> J. 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没有利益冲突的声明。

<html>在果蝇幼虫所描述的设置允许关联嗅觉学习的同等小学大脑内的调查。方法很简单，价格便宜，易于在实验室建立的，并不需要高科技设备9。我们提出了一个版本的实验中，研究食欲联想学习，加强果糖奖励11。所描述的设置是根据一系列的参数化的全面的调查研究，训练试验的数目的变化的，单一的检测与质量测定，保留时间，使用的气味和气味的浓度和性别9,15...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> CAS号 </td></tr><tr><td>果糖</td><td>西格玛</td><td> 47740 </td><td> 57-48-7 </td></tr><tr><td>氯化钠</td><td> Fluka公司</td><td> 71350 </td><td> 7647-14-5 </td></tr><tr><td>琼脂糖</td><td>西格玛</td><td> A5093 </td><td> 9012-36-6 </td></tr><tr><td> 1 - 辛醇</td><td>西格玛</td><td> 12012 </td><td> 111-87-5 </td></tr><tr><td>乙酸戊酯</td><td>西格玛</td><td> 46022 </td><td> 628-63-7 </td></tr><tr><td>灯油</td><td>西格玛</td><td> 18512 </td><td> 8012-95-1 </td></tr></tbody></table>

appetitive associative olfactory learning in drosophila larvae

在下面，我们将介绍的食欲联想在果蝇嗅觉学习方法的细节。的设置，与遗传干扰的组合，提供了一个手柄来分析在一个简单的幼虫脑特异性关联学习的神经细胞和分子的基本面。 生物可以用过去的经验来调整现在的行为。这种行为的潜在的收购可以定义为学习，这些记忆痕迹的潜力，1-4的物理基础。神经科学家试图了解这些过程是如何组织的分子和神经元在大脑中的变化，通过使用各种方法模式生物，从昆虫到脊椎动物5,6。对于这样的努力是有帮助的使用是简单和实验模型系统，。 果蝇幼虫已被证明是满足这些需求的基础上提供强大的行为分析，存在的各种转基因技术的基本组织的神经系统，包括约10000个神经元（虽然与一些让步:认知的限制，一些行为的选择，和丰富的经验可疑）7-10 。 果蝇幼虫可以形成协会气味和食欲的味觉增强，如糖​​11-14之间。在一个标准的检测，在实验室中成立B.格柏，动物收到两气味的倒数培训:第一组的幼虫接触到的气味，连同与味觉增强剂（白糖奖励），随后接触到的气味B没有加固9。同时接收倒数第二组的幼虫经历气味A的训练，而没有加固，随后暴露于气味乙加固（糖的奖励）。在下面的两个群体是工商业污水附加费特德自己的喜好两者之间的气味。相对较高的奖励气味的偏好反映联想学习 - 的性能指数（PI）。有关的各项性能指标的关联性的结论是令人信服的，因为除了从应急的气味和促味剂，其它参数，如气味和奖励曝光，通过时间和处理没有显着差异，两组9。

图1A示出了概述幼虫嗅觉联想学习的实验程序。通过配对两个气味之一与糖奖励幼虫获得表达朝向比较风范气味的奖励的气味中的一个有吸引力的响应潜在的行为。两组的幼虫始终的培训，通过配对的气味OCT或AM的增强剂。性能指数（PI）测量的相关功能之间的相互培训的群体偏好的差异。 在相关功能进行了分析，在转基因幼虫，基本感觉运动能力的测试是必需?...

Watch this Scientific Journal Video about Appetitive Associative Olfactory Learning in Drosophila Larvae at JoVE.com

Appetitive Associative Olfactory Learning in Drosophila Larvae

果蝇幼虫能联想到刺激气味，味觉奖励。在这里，我们描述了一个简单的行为范式，使食欲关联嗅觉学习分析。

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 ...

appetitive-associative-olfactory-learning-in-drosophila-larvae

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JoVE Journal

Neuroscience

Appetitive Associative Olfactory Learning in Drosophila Larvae

18.8K 次观看.  University of Konstanz. 果蝇幼虫能联想到刺激气味，味觉奖励。在这里，我们描述了一个简单的行为范式，使食欲关联嗅觉学习分析。

视频: Appetitive Associative Olfactory Learning in Drosophila Larvae

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

线虫的积极丁酮学习，短期，长期联想记忆检测

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

科研

神经科学

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 ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

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 ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

小鼠嗅觉学习和鉴别的客观和可重复性试验

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.