资助这项研究是由一个程序从佛罗里达州立大学医学院和生物科学系于前苏联的支持提供了神经科学奖。额外的经费是由格兰特在急救从酒精饮料制造商的研究基金提供的。

在平衡翼1。大会基本原理和概况：该系统的设计管理酒精蒸气的控制百分比苍蝇。注意： 图1提供了一示意性的概述的平衡杆设置为三个阶段（空气流的组件，设置在醇和水的瓶子，并观察小瓶组件）下面描述。总之，一个稳定的气流被分成通过酒精和水，鼓泡分别混合，给药于4观察小瓶两个馏分。 <ol><li>气流大会<ol><li>连接一段简短的弹性硅胶管连接到无论是建筑物空调或水族箱增氧机来生成使用Y型连接器的稳定的空气流量和分流。连接所述第一分支的空气流量调节器，用于控制空气的总量通过系统（通常千毫升/分钟4观察小瓶）。 </li><li>插入快速连接器在所述第二管使空气流可在实验开始时不影响校准的气流被中断。添加一个Y形连接器和各分支管连接到气流调节器。 </li><li>连接管的气流调节器，然后连接两个气流米。 </li></ol></li><li>建立了酒精和水的瓶<ol><li>加柔性管来的气流米的出口，然后将细玻璃管（1毫升玻璃移液管部分）与90°弯曲成管的每个长度的末端。这将作为进气空气流进入水和酒精瓶。 </li><li>将橡胶塞与2个孔，通过它们放入盛有酒精和水的瓶子。保持两个瓶在恒温下2℃比使用水浴周围空气的温度更高。在我们的实验中，对环境的室温保持在25℃，同时在水浴是在27℃下</li><li>将直玻璃吸管的一IR入口穿过橡胶塞和延伸进入所述流体，直至从瓶的底部约1厘米。 </li><li>插入的玻璃移液管肘节成在橡胶塞的剩余孔中，直到该玻璃的端部是与瓶内的止动器的底部齐平。插入管子的另一长度这个出气口。 </li><li>使用Y型连接器团聚空气流，并使用硅胶管的另一长度，通过一个空混合瓶或瓶子通过双孔橡皮塞插入弯曲的玻璃吸管部分，以​​引导气流。使用硅胶管的另一长度为出口混合气流。 </li></ol></li><li>观察瓶的大会<ol><li>分割的出口气流从混合箱2-3X新兴取得空气的4或8个更小的流，使多个观测小瓶可以在每个实验中被使用。将柔性硅胶管，以观察小瓶。 </li><li>设置observati使用用橡胶塞含两个孔，通过该玻璃管提供一种用于酒精蒸汽入口和一个出口的密封空小瓶小瓶中。 </li><li>覆盖网的第一玻璃管的端部，用一小片软塑料管保持丝网到位。通过第一孔插入该管，直到它延伸到所述小瓶的大约一半的长度。如果需要，使用铁氟龙胶带，以获得舒适的位置。 </li><li>插入第二玻璃管也与端部覆盖扣除，直到它与橡胶塞的内边缘平齐。 </li><li>水平放置在一个白色纸条的小瓶以最大化与红色的昏暗光线条件下的苍蝇对比度。 </li><li>气流的混合适当的馏分通入酒精和水，通过气流冒泡。连续监测的空气压力，并根据需要来维持空气流的期望的混合进行调整。 注：连续运行几个飞律师分析在一个小房间并行，甚至一个单一的检测可导致酒精蒸气的明显积累。为了避免酒精蒸气的潜在影响，研究者在一个封闭的房间中不断释放，相应的系统需要到位的充分消除了实验过程中产生酒精蒸气。要删除的酒精蒸气，连接6-12英寸的管件上的第二玻璃管从每个小瓶突出，捆绑他们，并直接向漏斗真空系统。研究人员也应确保实验测试房间充分通风。 </li></ol></li></ol> 2，准备实验动物基本原理和概况：适当的文化和苍蝇的住房将减少变异中的数据。这是通过标准化的应力经历了苍蝇和最小化来实现。出于这个原因，没有麻醉（CO 2或替代品）用于杜里纳克任何协议的步骤如下。此外，苍蝇应该是年龄匹配的整个实验时间点为是标准的其他行为的分析，包括学习和记忆的实验17，尽量减少可变性。 不同的光：暗光条件下可以用来探测生物钟在乙醇的行为反应的功能。如果要判断一个昼夜节律存在，可以实验下一个定义LD周期内执行特定Zeitgeber时报（ZT）来衡量性能。 ZT 0代表黎明和被定义为下LD循环灯的时间，而ZT 12是时候关灯用12:12小时LD周期。在恒定的条件下，昼夜时间（CT）测量时间为动物在没有环境信号， 即自由运行时间，并且是与前面的LD夹带周期。在野生型果蝇 ，CT反映的是前ZT第几个天恒定的条件下作为自由运行的昼夜周期和节奏s为〜24小时。为了测量昼夜调制和消除行为急性光效，苍蝇夹带光：暗循环，然后转移至恒暗光条件下（日）之前的实验。生理实验对DD的第二天进行特定的昼夜时间（CT）来衡量绩效。 在果蝇中，连续光（LL）条件下导致生理功能障碍的核心昼夜节律基因和破坏行为的昼夜节律就证明了心律失常自发活动18-21和心律失常短期记忆17湿布或废止分子振荡。该协议被优化用于昼夜节律的研究，并且可以简化用于其它实验。红色的小灯在12英寸的管道使用;所有生理实验使用的是昏暗的红光（环境头顶红灯&lt;1勒克斯的台式进行〜1勒克斯光）。 <ol><li>在25°C下12点12分小时光照后的苍蝇：黑暗夹带条件（LD）。 </li><li>收集新鲜eclosed苍蝇在第1天的日光期末，并将它们存储为LD条件下24小时的控股含有少量的高浓度琼脂食物，尽量减少食物粘性小瓶。注：为了确保健康，发育正常果蝇收集，只能用苍蝇在第一天收集羽化后开始在培养瓶。 </li><li>收集约30（25-35）批次苍蝇使用吸气第2天向光期结束，并转移到新的控股瓶。 </li><li>使用强光源直接苍蝇小瓶的尽头。在此范围内，如行为观察的报告形式与苍蝇在每个实验结束时计数的总数的百分比苍蝇在每个小瓶中的确切数目并不重要。 </li><li>保持苍蝇DD条件下，在25°C FOS两个天。 </li><li>在实验当天，将收纳在不同的条件或孵化比实验行为室在室内放置至少1小时，在实验之前，其他所有的苍蝇。驯化减少了可变性，由于温度变化或湿度。 </li><li>为了测试行为的昼夜调制使得在一天（CT 1，5，9，13，17和21）六个时间点的观测。 </li><li>在单一组的行为学实验，增加实验设计的鲁棒性和可变性的具体最小化到一个单一的实验比较多的时间点。例如，CT 1和CT 13观测可同时获得如果两个孵化器具有相对的光 - 暗时间表用于夹带。 注：以上介绍的准备实验动物在不断的黑暗昼夜条件下进行试验。不同的光：暗光条件下可以用来探测时钟的功能在行为反应的醇。如果要判断一个昼夜节律存在的实验可以在一个周期LD在特定Zeitgeber时报（ZT）进行衡量绩效。此外，该协议可以用于在恒定光照条件下进行实验测试生理功能障碍引发苍蝇。对于测试苍蝇装在光照条件下的替代方案，行为分析仍应黑暗条件下进行。苍蝇应该转移到黑暗1小时的实验之前，对行为的变异减少因光线对行为的急性影响。 </li></ol> 3，行为观察基本原理和概况：以下酒精的给药方案进行了优化昏暗的红光条件下的观察。这两种行为的措施翻正反射消失和镇静代表飞醉的两个不同的点。 LORR代表解酒的后期结合点的损失Øf电机和姿势控制，中毒而镇静措施很晚终点。基因型或生理调节可能会影响这两项措施有所不同;因此人们可能希望同时检查。总之，苍蝇在小瓶加载，蝇显示LORR或镇静的数量进行评分中的酒精蒸气曝光每隔5分钟，和苍蝇在实验结束时计算的总数。 <ol><li>在实验开始前，通过系统（通过水和酒精瓶空气鼓泡）下至少10分钟运行的空气，并使用该时间来校准气流。 </li><li>断开快速释放停止气流。加载苍蝇在小瓶中，并重新气流并启动定时器。注意：如果反应迟钝或苍蝇死苍蝇留在控股小瓶，这可能是反映应力条件。在一般情况下，这些条件可以由容纳在所述保持小瓶苍蝇更少或使用轻微减少食物的粘性可以减轻食物制备过程中LY更高的琼脂浓度。为了获得最佳的行​​为分析和实验之间的最小变异，苍蝇应该是之前的实验健康。 </li><li>为了获得准确的计时，使用一个定时器来保持酒精暴露的总时间的轨道，并使用第二计数计时器回为纪念5分钟的间隔。 </li><li>下方放置小瓶一张白纸可以提高对比度，飞的知名度，尤其是在昏暗的红灯条件。 </li><li>定期检查气流实验过程中保持恒定的水平。通常，一旦气流稳定后，它们在实验的长度期间保持稳定。 </li><li>计数的苍蝇已失去正位反射一次，每5分钟进行1小时内的数目。作为醇敏感性的基因型和遗传背景之间变化，这可能是可取的，以执行更频繁的评估或作实验的时间较长。 </li><li>将样品瓶slightlY从表面上看，和直接从红光手电朝着小瓶后面的纸张的光。保持手持红色手电筒在该实验瓶中的距离至少为12英寸到维持清淡水平不低于1勒克斯更大的暗淡红光条件下的所有实验。 </li><li>使用测光表来建立所有实验的标准测量光照水平。 </li><li>确定苍蝇已失去翻正反射施加自来水公司向小瓶，并计算有多少苍蝇未能在大约4秒合适自己的号码。这显示LORR苍蝇可能仍然把他们的腿和翅膀，但不能把自己的正直。 </li><li>在会议结束时，计数的苍蝇在每个小瓶的总数。 注意：有时，单个苍蝇可能装载时飞入试验小瓶制动器和侧之间被捕获。因为这是在黑暗中进行，它可能不容易发现，所以有必要计算总麻木蝇呃在实验过程中正确计算百分比的末端。 </li></ol>此外，该程序也可被用来测量镇静苍蝇，它代表一个不同的行为端点。而镇静剂苍蝇失去了正确的反射，镇静需要更大的酒精暴露。行为上，可以镇静的特点是完全没有明显的运动功能具有以下AA自来水公司向小瓶苍蝇一动也不动在小瓶中。镇静，计数苍蝇依然没有腿挥舞着小瓶的坚定水龙头下面一动不动交付的数量。此外，小瓶可以卷起一边到另一边，以确定个别苍蝇是否仍保留其敛反射。 4，数据分析<ol><li>确定百分比LORR在每个基于苍蝇在每个小瓶中的总数评估的时间。 </li><li>通过calculati昼夜的时间点或品系间的差异估算纳克的50％LORR对于每个样品，它落在S形曲线的线性部分内（见下面的图2）。 </li><li>另类统计： <ol><li>如果计划的基因型之间的比较，希望采用重复测量ANOVAand确定的时间点的范围内，该差异与事后测试显著（ 图3）分析整个时间过程。对于这些测试，我们优选使用0.001的值。这允许在单个曝光时间的差异进行评估，以及在该曲线的斜率基因型之间的差异。 </li><li>在对灵敏度的差异可为酒精暴露于如从图中（ 图4）的直线部分的镇静特定响应的特定的时间来确定。 </li><li>菌株或昼夜时间点之间的差可以使用标准的F统计量和事后测试来估计。 </li></ol></li></ol>

<ol>
	<li>Linde, K., Lyons, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+acute+alcohol+sensitivity+but+not+acute+tolerance+in+Drosophila.">Circadian modulation of acute alcohol sensitivity but not acute tolerance in Drosophila.</a> Chronobiol. Int. 28, 397-406 (2011).</li><li>Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., Heberlein, U. <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+alcohol+reward.">A Drosophila model for alcohol reward.</a> Nat Neurosci. 14, 612-619 (2011).</li><li>Shohat-Ophir, G., Kaun, K. R., Azanchi, R., Mohammed, H., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+deprivation+increases+ethanol+intake+in+Drosophila.">Sexual deprivation increases ethanol intake in Drosophila.</a> Science. 335, 1351-1355 (2012).</li><li>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=The+fruit+fly:+A+model+organism+to+study+the+genetics+of+alcohol+abuse+and+addiction.">The fruit fly: A model organism to study the genetics of alcohol abuse and addiction.</a> Cell. 93, 909-912 (1998).</li><li>Guarnieri, D. J., Heberlein, U. <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,+a+genetic+model+system+for+alcohol+research.">Drosophila melanogaster, a genetic model system for alcohol research.</a> International Review of Neurobiology. 54, 203-232 (2003).</li><li>Scholz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intoxicated+fly+brains:+Neurons+mediating+ethanol-induced+behaviors.">Intoxicated fly brains: Neurons mediating ethanol-induced behaviors.</a> J. Neurogenet. 23, 111-119 (2009).</li><li>Wolf, F. W., Rodan, A. R., Tsai, L. T. Y., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+analysis+of+ethanol-induced+locomotor+stimulation+in+Drosophila.">High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila.</a> J. Neurosci. 22, 11035-11044 (2002).</li><li>Schumann, G., Spanagel, R., Mann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidate+genes+for+alcohol+dependence:+Animal+studies.">Candidate genes for alcohol dependence: Animal studies.</a> Alcoholism: Clinical and Experimental Research. 27, 880-888 (2003).</li><li>Singh, C. M., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+acute+ethanol-induced+behaviors+in+Drosophila.">Genetic control of acute ethanol-induced behaviors in Drosophila.</a> Alcohol Clin Exp Res. 24, 1127-1136 (2000).</li><li>Perreau-Lenz, S., Zghoul, T., de Fonseca, F. R., Spanagel, R., Bilbao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+regulation+of+central+ethanol+sensitivity+by+the+mPer2+gene.">Circadian regulation of central ethanol sensitivity by the mPer2 gene.</a> Addiction Biology. 14, 253-259 (2009).</li><li>Brager, A. J., Prosser, R. A., Glass, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+acamprosate+modulation+of+elevated+ethanol+drinking+in+mPer2+clock+gene+mutant+mice.">Circadian and acamprosate modulation of elevated ethanol drinking in mPer2 clock gene mutant mice.</a> Chronobiol. Int. 28, 664-672 (2011).</li><li>Sinclair, J. D., Geller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+consumption+by+rats+under+different+lighting+conditions.">Ethanol consumption by rats under different lighting conditions.</a> Science. 175, 1143-1144 (1972).</li><li>Danel, T., Jeanson, R., Touitou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+pattern+in+consumption+of+the+first+drink+of+the+day+in+alcohol-dependent+persons.">Temporal pattern in consumption of the first drink of the day in alcohol-dependent persons.</a> Chronobiol. Int. 20, 1093-1102 (2003).</li><li>Kapfhamer, 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=Taok2+controls+behavioral+response+to+ethanol+in+mice.">Taok2 controls behavioral response to ethanol in mice.</a> Genes, brain, and behavior. 12 (1), 87-97 (2012).</li><li>Lasek, A. 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=An+evolutionary+conserved+role+for+anaplastic+lymphoma+kinase+in+behavioral+responses+to+ethanol.">An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol.</a> PLoS One. 6, 226-236 (2011).</li><li>Lasek, A. W., Giorgetti, F., Berger, K. H., Tayor, S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmo+genes+regulate+behavioral+responses+to+ethanol+in+Drosophila+melanogaster+and+the+mouse.">Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse.</a> Alcohol Clin Exp Res. 35, 1600-1606 (2011).</li><li>Lyons, L. C., Roman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+short-term+memory+in+Drosophila.">Circadian modulation of short-term memory in Drosophila.</a> Learning &amp; Memory. 16, 19-27 (2009).</li><li>Hamblen-Coyle, M. J., Wheeler, D. A., Rutila, J. E., Rosbash, M., 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=Behavior+of+period-altered+circadian-rhythm+mutants+of+Drosophila+in+ligh-dark+cycles+(Diptera+Drosophilidae).">Behavior of period-altered circadian-rhythm mutants of Drosophila in ligh-dark cycles (Diptera Drosophilidae).</a> J. Insect Behav. 5, 417-446 (1992).</li><li>Konopka, R. J., Pittendrigh, C., Orr, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+behavior+associated+with+altered+homeostasis+and+photosensitivity+of+Drosophila+clock+mutants.">Reciprocal behavior associated with altered homeostasis and photosensitivity of Drosophila clock mutants.</a> J. Neurogenet. 6, 1-10 (1989).</li><li>Power, J. M., Ringo, J. M., Dowse, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+period+mutations+and+light+on+the+activity+rhythms+of+Drosophila+melanogaster.">The effects of period mutations and light on the activity rhythms of Drosophila melanogaster.</a> Journal of Biological Rhythms. 10, 267-280 (1995).</li><li>Yoshii, 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=Temperature+cycles+drive+Drosophila+circadian+oscillation+in+constant+light+that+otherwise+induces+behavioural+arrhythmicity.">Temperature cycles drive Drosophila circadian oscillation in constant light that otherwise induces behavioural arrhythmicity.</a> Eur. J. Neurosci. 22, 1176-1184 (2005).</li><li>Berger, K. H., Heberlein, U., Moore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+chronic:+two+distinct+forms+of+ethanol+tolerance+in+Drosophila.">Rapid and chronic: two distinct forms of ethanol tolerance in Drosophila.</a> Alcohol Clin Exp Res. 28, 1469-1480 (2004).</li><li>Scholz, H., Ramond, J., Singh, C. M., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+ethanol+tolerance+in+Drosophila.">Functional ethanol tolerance in Drosophila.</a> Neuron. 28, 261-271 (2000).</li><li>Kong, E. 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=Ethanol-regulated+genes+that+contribute+to+ethanol+sensitivity+and+rapid+tolerance+in+Drosophila.">Ethanol-regulated genes that contribute to ethanol sensitivity and rapid tolerance in Drosophila.</a> Alcohol Clin Exp Res. 34, 302-316 (2010).</li><li>Borycz, J., Borycz, J., Kubow, A., Lloyd, V., Meinertzhagen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+ABC+transporter+mutants+white,+brown+and+scarlet+have+altered+contents+and+distribution+of+biogenic+amines+in+the+brain.">Drosophila ABC transporter mutants white, brown and scarlet have altered contents and distribution of biogenic amines in the brain.</a> J. Exp. Biol. 211, 3454-3466 (2008).</li><li>Sitaraman, 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=Serotonin+is+necessary+for+place+memory+in+Drosophila.">Serotonin is necessary for place memory in Drosophila.</a> Proceedings of the National Academy of Sciences. 105, 5579-5584 (2008).</li><li>Bainton, R. J., 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=Dopamine+modulates+acute+responses+to+cocaine,+nicotine+and+ethanol+in+Drosophila.">Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila.</a> Current Biology. 10, 187-194 (2000).</li><li>Kong, E. 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=A+pair+of+dopamine+neurons+target+the+D1-like+dopamine+receptor+DopR+in+the+central+complex+to+promote+ethanol-stimulated+locomotion+in+Drosophila.">A pair of dopamine neurons target the D1-like dopamine receptor DopR in the central complex to promote ethanol-stimulated locomotion in Drosophila.</a> Plos One. 5, (2010).</li><li>Xu, J., Kochanek, K. D., Murphy, S. L., Tejada-Vera, 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> Deaths: Final data for 2007. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Center+on+Addiction+and+Substance+Abuse.">The National Center on Addiction and Substance Abuse.</a> Shoveling up II: The impact of substance abuse on federal, state and local budgets. , (2009).</li><li>NIAAA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+economic+costs+of+alcohol+abuse+in+the+United+States.">Estimated economic costs of alcohol abuse in the United States.</a> , (1992).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+ethanol+consumption+in+Drosophila+models+features+of+addiction.">Preferential ethanol consumption in Drosophila models features of addiction.</a> Current Biology. 19, 2126-2132 (2009).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addiction-like+behavior+in+Drosophila.">Addiction-like behavior in Drosophila.</a> Communicative &amp; Integrative Biology. 3, 357-359 (2010).</li><li>Rodan, A. R., Rothenfluh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+behavioral+alcohol+responses+in+Drosophila.">The genetics of behavioral alcohol responses in Drosophila.</a> International Review of Neurobiology. 91, 25-51 (2010).</li><li>Boothroyd, C. E., Young, M. W., Pfaff, D. W., Kieffer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+Biophysical+Mechanisms+of+Arousal,+Alertness,+and+Attention.">Molecular and Biophysical Mechanisms of Arousal, Alertness, and Attention.</a> Annals of the New York Academy of Sciences. 1129, 350-357 (2008).</li><li>Nitabach, M. N., Taghert, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Drosophila+circadian+control+circuit.">Organization of the Drosophila circadian control circuit.</a> Current Biology. 18, 84-93 (2008).</li><li>Sheeba, V. <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+melanogaster+circadian+pacemaker+circuit.">The Drosophila melanogaster circadian pacemaker circuit.</a> J. Genet. 87, 485-493 (2008).</li><li>Cohan, F. M., Graf, J. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latitudinal+cline+in+Drosophila+melanogaster+for+knockdown+resistance+to+ethanol+fumes+and+for+rates+of+response+to+selection+for+further+resistance.">Latitudinal cline in Drosophila melanogaster for knockdown resistance to ethanol fumes and for rates of response to selection for further resistance.</a> Evolution. , 278-293 (1985).</li><li>Moore, M. 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=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93, 997-1007 (1998).</li><li>Berger, K. 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=Ethanol+sensitivity+and+tolerance+in+long-term+memory+mutants+of+Drosophila+melanogaster.">Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster.</a> Alcohol Clin Exp Res. 32, 895-908 (2008).</li><li>Pohl, J. 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=Circadian+Genes+Differentially+Affect+Tolerance+to+Ethanol.">Circadian Genes Differentially Affect Tolerance to Ethanol.</a> in Drosophila. Alcoholism: Clinical and Experimental Research. , (2013).</li><li>Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+evoked+locomotor+behavior+in+Drosophila+reveals+a+role+for+integrins+in+ethanol+sensitivity+and+rapid+ethanol+tolerance.">An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.</a> Alcoholism: Clinical and Experimental Research. 33, 1794-1805 (2009).</li><li>Rothenfluh, 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=Distinct+behavioral+responses+to+ethanol+are+regulated+by+alternate+RhoGAP18B+isoforms.">Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.</a> Cell. 127, (1016).</li></ol>

作者宣称，他们有没有竞争的财务权益。

酒精滥用和酒精中毒的社会成本是巨大的，无论是在人类29和经济成本方面30,31。 果蝇为模型提供了一个快速和灵活的系统可快速检查了大量个人的行为反应，因此已被广泛地用于这两种醇5,7,32-34和昼夜节律研究35-37。 在这里，我们描述了一个简单的协议，用于酒精蒸汽成蝇昼夜条件下的控制管理。 苍蝇在标准条件...

果蝇表现出双相的行为反应，以酒精1是类似于人类的反应，此药2,3。在初始接触低浓度的酒精，苍蝇展览增加自发活动，由于缺乏运动协调，姿势控制的损失和扶正反射（翻正反射消失的：翻正反射消失）取代，镇静（完全没有响应运动活动机械性刺激）作为暴露于酒精的进展4-9。内源性生物钟是一个强大酒精的敏感性和毒性的调制器在10,11小鼠，大鼠12观察，和人类13。在果蝇研究的最新进展表明，生物钟调节急性酒精的敏感性，但没有酒精的耐受1。通过突变体的研究和空间的转基因操作的强有力的遗传可在果蝇方法和时间上的基因表达提供了一个系统，允许在识别复杂行为的基本细胞和分子机制进展迅速。利用果蝇作为调查工具已经允许在了解酒精神经生物学实质性的进步，可以迅速转化为哺乳动物14-16。为了方便，通过该生物钟调节酒精的敏感性的分子机制的理解，并均匀地测量整个昼夜时间点的行为反应，适于在昏暗的红光条件下使用醇的给药方案是必需的。对于果蝇 ，酒精可通过食物补充对慢性接触或通过可靠的蒸汽急性暴露的形式，行政的酒精管理。在这里，我们描述了适用于失的扶正反射（翻正反射消失）1昼夜调制的评估，以及酒精管理协议镇静。 蝇夹带有12小时：12小时LD周期在恒定的温度和然后转移到控制光制度为2-5天取决于实验的问题。蝇中被称为平衡杆的装置暴露于乙醇蒸气。在该装置中，控制量的空气通过水和醇鼓泡;蒸气，然后混合并定向到小瓶外壳的苍蝇。每5苍蝇分钟被打进了失败，显示扶正反射或已成为镇静的数量。 LORR百分比为每个时间点计算并比较了昼夜时间点或苍蝇的菌株之间。酒精交付使用平衡翼酒精交付并结合行为分析选项的简便性和可靠性提供了黑暗的条件下进行昼夜实验一个显著的好处。

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Alcohol 190 proof</td>
			<td style="width:115px;">Various</td>
			<td style="width:119px;"></td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;"></td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:296px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Aerator</td>
			<td style="width:115px;">Local pet store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">We use Whisper 60</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Silicone tubing 1/8&#8221;</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">408060-0030</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">120&#176; Y Connector</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">82017-256</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Quick disconnects</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">46600-048</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:285px;">Plastic tube clamps</td>
			<td style="width:115px;">Bell-art products</td>
			<td style="width:119px;">132250000</td>
			<td style="width:296px;">Either this or next</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Miniature Air Regulator</td>
			<td style="width:115px;">McMaster-Carr</td>
			<td style="width:119px;">8727K11</td>
			<td style="width:296px;">Either this or previous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Miniature Air Regulator Mounting Bracket</td>
			<td style="width:115px;">McMaster-Carr</td>
			<td style="width:119px;">9891K66</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Gilmont size 12 flow meter</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">29895-242</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Tool clips</td>
			<td style="width:115px;">McMaster-Carr</td>
			<td style="width:119px;">1722A43</td>
			<td style="width:296px;">To hold flow meters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Vial</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">89092-722</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Rubber stopper with two holes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">59585-186</td>
			<td style="width:296px;">Fits in vials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">5 mm Pyrex Glass tubes</td>
			<td style="width:115px;">Trikinetics</td>
			<td style="width:119px;">PGT5x65</td>
			<td style="width:296px;">Fits best in previous stopper.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Teflon tape</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">To achieve snug fit in stoppers if necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Rubber stopper with two holes</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">59582-122</td>
			<td style="width:296px;">Fits our bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Disposable glass pipets</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">53283-768</td>
			<td style="width:296px;">Cut to length and bend by heating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Very fine nylon netting</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">Various</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">15 watt bulbs</td>
			<td style="width:115px;">Hardware store</td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Photographic red safe light filters</td>
			<td style="width:115px;"></td>
			<td style="width:119px;"></td>
			<td style="width:296px;">Overhead red light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Mini Flashlights with red filters</td>
			<td style="width:115px;">Mag-light</td>
			<td style="width:119px;"></td>
			<td style="width:296px;"></td>
		</tr>
	</tbody>
</table>

the flybar: administering alcohol to flies

果蝇（ 果蝇 ）是一种成熟的模式为酒精的研究和昼夜生物。最近，我们发现，生物钟调节酒精的敏感性，但不能耐受的形成。在这里，我们详细描述了我们的协议。酒精是给予使用平衡翼的苍蝇。在这种设置中，饱和酒精蒸气与加湿空气混合比例设置，并给予苍蝇在同时四根管子。苍蝇是为了尽量减少重复之间的差异在标准条件下饲养。三日龄果蝇不同的基因型或治疗方法，所用的实验中，优选通过匹配两个不同的时间点（ 例如 ，CT 5和CT 17）苍蝇进行直接比较成为可能。在实验过程中，苍蝇暴露1小时，以酒精蒸气的预定百分比和苍蝇的数量表现出翻正反射消失（LORR）的损失或sed的振动性进行计数，每5分钟。中的数据可以使用三种不同的统计方法来分析。首先是要确定在哪个苍蝇的50％已失去翻正反射时，使用方差分析（ANOVA）来确定的时间点之间是否存在显著差异。第二个是确定的百分比苍蝇指定的分钟数，后跟一个单因素方差分析后显示翻正反射消失。最后一种方法是分析整个时间序列采用多元统计。该协议也可以用于基因型之间的非生理实验或比较。

酒精的敏感性，使用的50％翻正反射消失为标志的昼夜调制。 一个典型的例子显示，白天昼夜调制在酒精的敏感性示于图2。LORR在进行DD在广州-S中的第 2天测量在六个时间点，50％翻正反射消失为每个时间点确定。分析表明，一天中的时间显著作用（ANOVA：F 5,45 = 7.39，P &lt;0.001，N = 6-10每个时间点）。费雪LSD检验表明CT1之间的显著差异与CT5，C...

Watch this Scientific Journal Video about The FlyBar: Administering Alcohol to Flies at JoVE.com

The FlyBar: Administering Alcohol to Flies

果蝇已经成为解剖的行为反应，以酒精的细胞和分子基础的一个显著的模型系统。在这里，我们提出了一个协议，用于酒精的敏感性数据的，可以很容易地应用到其他的实验，是非常适合本科生科研昼夜上下文的集合。

该平衡翼：管理对酒精苍蝇

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

the-flybar-administering-alcohol-to-flies

Research

JoVE Journal

Neuroscience

10.6K 次观看.  Florida State University.  果蝇已经成为解剖的行为反应，以酒精的细胞和分子基础的一个显著的模型系统。在这里，我们提出了一个协议，用于酒精的敏感性数据的，可以很容易地应用到其他的实验，是非常适合本科生科研昼夜上下文的集合。 

视频: The FlyBar: Administering Alcohol to Flies

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

一个简单的方法来衡量在果蝇的乙醇灵敏度

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

视频成像和时空地图来分析胃肠动力小鼠

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...