本研究描述了一种简单的行为范式，可用于分析成年果蝇的厌恶性关联学习。该方法基于特定环境背景与电击之间形成关联，从而抑制果蝇与生俱来的负向趋地性行为。
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摘要
摘要
本方案描述了一种用于分析成年果蝇（Drosophila melanogaster）厌恶性关联学习的新范式。该范式类似于实验啮齿类动物中的被动回避行为，即动物学会回避其先前曾遭受电击的区域。该检测方法利用了果蝇的负向趋地性，表现为当果蝇被置于垂直表面时具有向上攀爬的倾向。实验装置由上下两个垂直排列的隔室组成。在第一次试验中，将一只果蝇放入下方隔室，它通常在3–15秒内离开该隔室 ，进入上方隔室并在此处受到电击。在24小时后的第二次试验中，果蝇进入上方隔室的潜伏期显著延长。同时，与第一次试验相比，其受到的电击次数减少，表明果蝇已对上方隔室形成了长期记忆。潜伏期和电击次数的记录可使用计数器和秒表完成，也可借助基于Arduino的简易设备进行。为展示该检测方法的应用，本文对D. melanogaster和D. simulans的雄性和雌性果蝇的被动回避行为进行了表征。对潜伏期和电击次数的比较显示，D. melanogaster和D. simulans果蝇均能有效习得被动回避行为。雄性和雌性果蝇之间未观察到统计学上的显著差异。然而，在第一次试验中，雄性进入上方隔室的速度略快，而雌性在每次记忆保持试验中受到的电击次数略高。西方饮食（WD）显著损害了雄性果蝇的学习与记忆能力，而飞行锻炼可抵消这种不利影响。综上所述，果蝇的被动回避行为提供了一种简单且可重复的检测方法，可用于研究学习与记忆的基本机制。
引言
学习与记忆是一种在进化上古老的环境适应机制，从Drosophila (D.)到人类均高度保守1。果蝇是研究学习与记忆基本原理的有力模式生物，因其提供了丰富的强大遗传工具，可用于解析其内在的分子机制2。早期的遗传筛选研究利用了果蝇依赖敏锐嗅觉寻找食物、潜在配偶以及躲避天敌的特性6，通过嗅觉条件化方法鉴定出对学习与记忆至关重要的rutabaga3、amnesiac4和dunce5基因2。
由于Tully和Quinn引入了嗅觉T型迷宫7,8，嗅觉条件化已成为研究学习与记忆机制的常用范式。随后，其他用于检测多种类型学习与记忆的方法也被相继提出，包括视觉条件化9、求偶行为条件化10、厌恶性趋光抑制实验11以及寄生蜂暴露条件化12。然而，这些实验方法大多需要复杂的装置，必须在大学的工作坊中定制，或通过供应商购买。本文所描述的范式基于一种简单的行为学检测方法，用于研究果蝇的厌恶性关联学习，仅需少量易得的材料即可轻松组装完成。
所述范式相当于实验小鼠和大鼠中的被动（或抑制性）回避行为，即动物学会回避其先前曾遭受电击的区域13。在啮齿类动物中，该实验基于它们天生回避明亮光线并偏好较暗区域的行为特征14。在首次试验中，将动物置于明亮的区域，动物会迅速离开该区域，进入黑暗隔间，并在此处给予足部电击。通常，仅一次训练即可形成稳定的长期记忆，表现为24小时后潜伏期显著延长。该潜伏期随后被用作评估动物对厌恶刺激与特定环境之间关联记忆能力的指标15。
本研究描述了一种使用D.作为模型系统的类似实验方法，该模型相较于啮齿类动物模型具有多项优势，包括成本效益高、样本量大、无需监管审批以及可利用强大的遗传学工具16,17。该实验基于负向趋地性行为，即当果蝇被置于垂直表面时，会表现出向上攀爬的倾向18。实验装置由两个垂直的腔室组成。在首次试验中，将一只果蝇放入下方腔室，它通常在3–15秒内离开该区域，进入上方腔室并受到电击刺激。在持续1分钟的试验过程中，部分果蝇可能偶尔再次进入上腔室，从而受到额外电击。在24小时后的测试阶段，果蝇进入上腔室的潜伏期显著延长，同时受到的电击次数较第一天明显减少，表明果蝇已形成对上腔室的厌恶性关联记忆。通过分析潜伏期、电击次数以及理毛行为的持续时间和频率，可评估动物的行为表现及其建立并记忆厌恶刺激与特定环境之间关联的能力。代表性结果表明，西方饮食（WD）暴露显著损害了雄性果蝇的被动回避行为，提示WD对果蝇的行为和认知功能具有显著负面影响。相反，飞行锻炼可缓解WD的不利影响，改善果蝇的被动回避行为。
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方案
1. 被动回避装置的准备
- 在14 mL聚丙烯培养管的管壁表面，距离管底8 mm处，垂直钻一个4 mm的孔。
注意：使用电钻和5/32英寸钻头可获得最佳效果。 - 使用钢制美工刀，切下14 mL聚丙烯培养管的上半部分，保留45 mm长的管底片段。该片段作为下部室。
- 使用单刃剃须刀片切去1,000 µL蓝色吸头的尖端，使开口足够宽，以便单只果蝇通过。切去蓝色吸头的收缩部分，制成一段12 mm长的片段。将此片段牢固地插入下部室的4 mm孔中。该片段用作转移果蝇的装载口。
- 截取一段15 mm长、内径为5/8"的透明乙烯基软管（见材料表），作为连接套管。从相对两端将上部室和下部室插入该套管中，以牢固连接上下两室。
- 使用双爪可调夹具将组装好的装置固定在垂直支架上。将装置垂直放置，其中电击管作为上部室。
- 将电击管的导线连接至电刺激器（见材料表），以施加电击刺激。训练持续时间为1分钟。
注意：为便于观察，可在电击管后方放置一张白纸，作为装置的白色背景。在电击室上方放置一盏配有75 W等效柔光灯泡的灯具。在装置前方放置一个带可调臂的放大镜照明灯。被动回避装置的示意图见图1。
2. 被动回避实验前果蝇的准备
- 固定3-4日龄 D. melanogaster 或 D. simulans 使用冰块对果蝇进行冷麻醉，并在实验前24小时将其转移至含食物的单独小瓶中（每瓶1只果蝇），操作步骤参照先前所述方法19.
注意：此处描述的实验比较了3-4日龄雄性 对比雌性果蝇在 D. melanogaster 和 D. simulans. - 在行为学实验之前，对所有小瓶进行编码。为此，为每组分配一个字母，例如 "A", "B", "C"等，并为每只果蝇编号。在所有数据记录和分析完成之前，不得公开此编码。每种基因型或处理组至少使用20只果蝇，以减少个体差异的影响。
注意：进行实验与分析 "盲法" 可避免在评估果蝇行为表现及数据分析时引入偏差。
3. 进行第一次试验
- 使用果蝇口吸管（参见 材料表如前所述20, 轻轻地将一只果蝇从单独的试剂管转移到下层隔室 通过 装载平台。用口吸器轻轻吸气，吸取一只果蝇。通过轻轻向装载平台吹气，将果蝇释放。
注意：在抓取和装载动物时，应避免使其受到应激。 - 果蝇放入下层室后，立即启动1分钟计时器和秒表。
注意：使用秒表测量潜伏期，使用计数器统计电击次数。 - 按下秒表，当果蝇用四只爪子完全进入电击管时记录首次潜伏期。开启刺激器，对果蝇施加电刺激。刺激参数为：120伏，持续时间1000毫秒，脉冲频率1脉冲/秒（PPS），脉冲串持续时间2000毫秒。
- 如果果蝇重新进入电击管，则给予额外电击。使用计数器或基于Arduino的计数装置，在1分钟试验期间记录果蝇接受的电击次数（参见 材料表）。如果使用基于 Arduino 的计数器，请遵循以下步骤。
注意：可选的基于Arduino的设备AKM-007（参见 材料表可以使用该设备通过按压和释放相应按钮来测量每只动物的时间、潜伏期、电击次数以及理毛行为的频率和持续时间。设备上的按钮分别用于测量潜伏期、施加并记录电击次数，以及测量理毛行为的频率和持续时间。- 按压 开始 步骤 3.2 中的按钮，并按下 休克 步骤 3.3 中的按钮
- 要记录一次理毛行为的持续时间，请按下 梳理行为 在设备上开始理毛行为时按下按钮，并在理毛行为结束时松开该按钮。
注意：理毛行为持续时间在1分钟试验期内全程记录。过度理毛可能提示动物处于应激状态21,22基于Arduino的设备将所有数据以CSV文件格式保存至存储卡中。
- 1 分钟试验结束后，轻柔地将果蝇转移回单独的样品管中。记录潜伏期、接受电击的次数以及行为上的任何显著变化。
- 用70%乙醇清洁下部和震荡仓，再用无尘清洁纸巾擦拭（参见 材料表），并用吹风机吹干。对下一只果蝇重复该实验步骤。
- 行为学实验结束后，用水和无味清洁剂清洗下部隔室，再用70%乙醇擦拭下部隔室和电击隔室，并过夜风干。
4. 进行第二次试验
- 24小时后，通过重复上述步骤（步骤3）进行第二次试验。测试果蝇的顺序应与前一天相同。
5. 结果分析
- 针对每组实验动物，计算试验1和试验2的平均潜伏期、平均电击次数以及理毛行为的持续时间。对于两组间的比较，采用学生t检验；对于多组比较，采用方差分析（ANOVA），并使用Tukey检验进行事后分析23。
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结果
被动回避实验在 D. melanogaster （Canton-S）和 D. simulans实验比较了连续试验之间的潜伏期和接受电击的次数。最初，实验使用3-4日龄的雄性 D. melanogaster 果蝇。果蝇在温湿度可控的环境中，饲养于标准的Bloomington配方饲料上。 24 °C 在12小时光照-黑暗循环、70%湿度及受控种群密度条件下饲养。通过保持所有组别的繁殖条件一致来控制密度。每瓶中放入15只雄性和15只雌性，共同繁殖48小时 24 °C，70% 湿度，12 小时光照周期的条件下繁殖后代。果蝇的被动回避行为通过四次间隔 24 小时、每次持续 2 分钟的实验进行研究。所有实验均在每天同一时间进行。实验过程中，将一只果蝇从单独的玻璃小瓶中轻轻吸出，并转移至下方隔间 通过 一个装卸平台（图1。实验结果表明 D. melanogaster 能够成功学习并记忆被动回避行为。在第一次试验中，未经训练的果蝇平均在16秒内（16.15±2.64）进入上层区域，并经常...
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讨论
避免威胁性刺激是多种物种适应性行为的关键特征，从 C秀丽隐杆线虫 对人类32通常涉及逃避厌恶性事件的回避学习程序，是研究实验啮齿类动物学习与记忆过程的常用行为学任务。13 自20世纪70年代以来32在主动回避实验中，一个中性刺激或条件信号（CS）之后会跟随一个厌恶事件或非条件信号（US），动物通过执行特定的行为任务来学会避免该厌恶事件。在被动回避实验中，动物需要将先前受到惩罚的行为与特定的环境背景相关联，从而避免遭遇厌恶的US。33较长的保留测试潜伏期表明记忆较好，提示动物对训练经历形成了详细的表征13被动回避训练包含单次试验；然而，该任务习得背后的脑机制十分复杂，因为动物需要学习将多种信息关联起来，包括环境信息、空间位置信息以及厌恶刺激。13改变这些刺激可用来研究情节性和情境性记忆类型13
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披露
作者声明无利益冲突。
致谢
本研究部分由美国国立卫生研究院（NIH）R15ES029673（AKM）资助。
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 布卢明顿配方饲料 | 营养果蝇饲料（Nutri-Fly） | 66-112 | 可从加利福尼亚州圣地亚哥市的 Genesee Scientific Inc. 公司购买 |
| 1000 µL 蓝色吸头 | 费舍尔（Fisher） | NC9546243 | |
| 17 x 100 mm 14 mL 聚丙烯培养管 | VWR | 60818-689 | |
| 基于 Arduino 的自动控制模块 | 本实验室自制 | AKM-007 | 该设备为可选部件。完整说明、示意图、接线图及代码可在东卡罗来纳大学数字市场（ECU Digital Market）获取 - https://digitalmarket.ecu.edu/akmmodule |
| 双显示屏双通道 数字时钟/计时器 | Digi-Sense | AO-94440-10 | https://www.amazon.com/Cole-Parmer-AO-94440-10-Dual-Display-2-Channel-Jumbo-Digit/dp/B00PR0809G/ref=sr_1_5?dchild=1&keywords=Dual-Display+timer+jumbo&qid=1627660660&sr= 8-5#customerReviews |
| 电子指针计数器 | 无（N/A） | 无（N/A） | https://www.amazon.com/gp/product/B01M8IRK6F/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1 |
| 费舍尔品牌 Sparkleen 1 型洗涤剂 | 费舍尔科技（Fisher Scientific） | 04-320-4 | |
| 果蝇口吸器 | 本实验室自制 | 制备方法见参考文献19 | |
| Grass S88 刺激器 | 无（N/A） | 无（N/A） | 可用任何能提供所述参数的刺激器替代 |
| Kim-wipes 擦拭纸 | 费舍尔科技（Fisher Scientific） | 06-666 | Kimberly-Clark 专业系列 34120 |
| 果蝇固定用金属块 | 本实验室自制 | 尺寸为 4 x 13 x 23.5 cm 的铝块 | |
| Nutiva 美国农业部（USDA）认证有机、非转基因红棕榈油 | Nutiva | 无（N/A） | https://www.amazon.com/Nutiva-Certified-Cold-Filtered-Unrefined-Ecuadorian/dp/B00JJ1E83G/ref=sxts_rp_s1_0?cv_ct_cx=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&dchild=1&keywords=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&pd_rd_i=B00JJ1E83G&pd_ rd_r=f35e9d2f-afe4-44b6-afc2-1c9cd705be18&pd_rd_w= R3Zb4&pd_rd_wg=eUv1m&pf_rd_ p=c6bde456-f877-4246-800f-44405f638777&pf _rd_r=M94N11RC7NH333EMJ66Y &psc=1&qid=1627661533&sr=1-1-f0029781-b79b-4b60-9cb0-eeda4dea34d6 |
| 电击管 | CelExplorer | TMA-201 | https://www.celexplorer.com/product_detail.asp?id=217&MainType=110&SubType=8 |
| 秒表 | Accusplit | A601XLN | https://www.amazon.com/gp/product/B0007ZGZYI/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1 |
| 透明乙烯基软管（外径 3/4”，内径 5/8”） | Lowes | 可从 Lowes 公司购买 |
参考文献
- Kandel, E. R., Dudai, Y., Mayford, M. R. The molecular and systems biology of memory. Cell. 157 (1), 163-186 (2014).
- McGuire, S. E., Deshazer, M., Davis, R. L. Thirty years of olfactory learning and memory research in Drosophila melanogaster. Progress in Neurobiology. 76 (5), 328-347 (2005).
- Livingstone, M. S., Sziber, P. P., Quinn, W. G. Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. Cell. 37 (1), 205-215 (1984).
- Quinn, W. G., Sziber, P. P., Booker, R. The Drosophila memory mutant amnesiac. Nature. 277 (5693), 212-214 (1979).
- Dudai, Y., Jan, Y. N., Byers, D., Quinn, W. G., Benzer, S. dunce, a mutant of Drosophila deficient in learning. Proceedings of the National Academy of Sciences of the United States of America. 73 (5), 1684-1688 (1976).
- Busto, G. U., Cervantes-Sandoval, I., Davis, R. L. Olfactory learning in Drosophila. Physiology. 25 (6), Bethesda, Md. 338-346 (2010).
- Tully, T., Quinn, W. G. Classical conditioning and retention in normal and mutant Drosophila melanogaster. Journal of Comparative Physiology. A: Sensory, Neural, and Behavioral Physiology. 157 (2), 263-277 (1985).
- Wright, N. J. Evolution of the techniques used in studying associative olfactory learning and memory in adult Drosophila in vivo: A historical and technical perspective. Invertebrate Neuroscience. 14 (1), 1-11 (2014).
- Vogt, K., Yarali, A., Tanimoto, H. Reversing stimulus timing in visual conditioning leads to memories with opposite valence in Drosophila. PloS One. 10 (10), 0139797(2015).
- Koemans, T. S., et al. Drosophila courtship conditioning as a measure of learning and memory. Journal of Visualized Experiments. (124), e55808(2017).
- Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration. Journal of Visualized Experiments. (49), e2504(2011).
- Bozler, J., et al. A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes. Plos Genetics. 13 (10), 1007054(2017).
- Atucha, E., Roozendaal, B. The inhibitory avoidance discrimination task to investigate accuracy of memory. Frontiers in Behavioral Neuroscience. 9, 60(2015).
- Thiels, E., Hoffman, E. K., Gorin, M. B. A reliable behavioral assay for the assessment of sustained photophobia in mice. Current Eye Research. 33 (5), 483-491 (2008).
- Detrait, E. R., Hanon, E., Dardenne, B., Lamberty, Y. The inhibitory avoidance test optimized for discovery of cognitive enhancers. Behavior Research Methods. 41 (3), 805-811 (2009).
- Piper, M. D. W., Partridge, L. Drosophila as a model for ageing. Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (9), Pt A 2707-2717 (2018).
- Chalmers, J., et al. A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes. Molecular Metabolism. 43, 101127(2021).
- Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Experimental Gerontology. 40 (5), 386-395 (2005).
- Yang, D. Simple homemade tools to handle fruit flies-Drosophila melanogaster. Journal of Visualized Experiments. (149), e59613(2019).
- Barradale, F., Sinha, K., Lebestky, T. Quantification of Drosophila grooming behavior. Journal of Visualized Experiments. (125), e55231(2017).
- Denmark, A., et al. The effects of chronic social defeat stress on mouse self-grooming behavior and its patterning. Behavioural Brain Research. 208 (2), 553-559 (2010).
- Kalueff, A. V., et al. Neurobiology of rodent self-grooming and its value for translational neuroscience. Nature Reviews: Neuroscience. 17 (1), 45-59 (2016).
- Motulsky, H. Intuitive biostatistics: A nonmathematical guide to statistical thinking. Fourth edition. , Oxford University Press. (2018).
- Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M., Syed, S. Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier. Elife. 7, 34497(2018).
- Mu, M. D., et al. A limbic circuitry involved in emotional stress-induced grooming. Nature Communications. 11 (1), 2261(2020).
- Song, C., Berridge, K. C., Kalueff, A. V. Stressing' rodent self-grooming for neuroscience research. Nature Reviews: Neuroscience. 17 (9), 591(2016).
- Wang, C., Chan, J. S., Ren, L., Yan, J. H. Obesity reduces cognitive and motor functions across the lifespan. Neural Plasticity. 2016, 2473081(2016).
- Lewis, A. R., Singh, S., Youssef, F. F. Cafeteria-diet induced obesity results in impaired cognitive functioning in a rodent model. Heliyon. 5 (3), 01412(2019).
- Yohn, S. E., Galbraith, J., Calipari, E. S., Conn, P. J. Shared behavioral and neurocircuitry disruptions in drug addiction, obesity, and binge eating disorder: Focus on Group I mGluRs in the mesolimbic dopamine pathway. ACS Chemical Neuroscience. 10 (5), 2125-2143 (2019).
- Lopez-Taboada, I., Gonzalez-Pardo, H., Conejo, N. M. Western Diet: Implications for brain function and behavior. Frontiers in Psychololgy. 11, 564413(2020).
- Murashov, A. K., et al. Preference and detrimental effects of high fat, sugar, and salt diet in wild-caught Drosophila simulans are reversed by flight exercise. FASEB Bioadvances. 3 (1), 49-64 (2021).
- Krypotos, A. M., Effting, M., Kindt, M., Beckers, T. Avoidance learning: A review of theoretical models and recent developments. Frontiers in Behavioral Neuroscience. 9, 189(2015).
- Encyclopedia of Neuroscience. Binder, M. D., Hirokawa, N., Windhorst, U. , Springer Berlin Heidelberg. 3093(2009).
- Mery, F., Belay, A. T., So, A. K., Sokolowski, M. B., Kawecki, T. J. Natural polymorphism affecting learning and memory in Drosophila. Proceedings of the National Academy of Sciences of the United States of America. 104 (32), 13051-13055 (2007).
- Tan, Y., Yu, D., Pletting, J., Davis, R. L. Gilgamesh is required for rutabaga-independent olfactory learning in Drosophila. Neuron. 67 (5), 810-820 (2010).
- Ögren, S. O., Stiedl, O. Encyclopedia of Psychopharmacology. Stolerman, I. P. , Springer Berlin Heidelberg. 960-967 (2010).
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重印与许可
勘误
Formal Correction: Erratum: Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning
Posted by JoVE Editors on 2/23/2022. Citeable Link.
An erratum was issued for: Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning. The Representative Results and Discussion sections were updated.
In the Representative Results, the legend for Figure 5 was updated from:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A) Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable increase in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P<0.05. One-way ANOVA with Tukey's multiple comparisons test.
to:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A) Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable decrease in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P<0.05. One-way ANOVA with Tukey's multiple comparisons test.
In the Discussion, the third paragraph was updated from:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if naïve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females' failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly higher in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
to:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if naïve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females' failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly lower in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
