JoVE Logo
发表
联系我们

登录

需要订阅 JoVE 才能查看此. 登录或开始免费试用。

本文内容

  • Erratum Notice
  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • Erratum
  • 转载和许可

Erratum Notice

Important: There has been an erratum issued for this article. Read More ...

摘要

这项工作描述了一个简单的行为范式，可以分析成年果蝇的厌恶联想学习。该方法基于抑制由于特定环境环境与电击之间形成的关联而导致的先天负地理出租车行为。

摘要

该协议描述了一种分析成年果蝇（Drosophila melanogaster）中厌恶性联想学习的新范例。这种范式类似于实验室啮齿动物的被动回避行为，其中动物学会避开以前接受过电击的隔间。该测定利用了苍蝇中的负地理出租车，这表现为当它们被放置在垂直表面上时爬上去的冲动。该设置由垂直方向的上部和下部隔间组成。在第一次试验中，一只苍蝇被放置在较低的隔间中，通常从那里在3-15秒内离开，然后进入上层隔间，在那里它受到电击。在第二次试验期间，24小时后，潜伏期显着增加。同时，与第一次试验相比，电击次数减少，表明苍蝇形成了关于上部隔室的长期记忆。延迟和冲击次数的记录可以使用计数计数器和秒表或基于Arduino的简单设备执行。为了说明如何使用该测定，本文表征了 D. melanogaster 和 D. simulans 男性和女性的被动回避行为。对潜伏期和冲击次数的比较表明， D. melanogaster 和 D. simulans 苍蝇都有效地学习了被动回避行为。雄性和雌性苍蝇之间没有观察到统计学差异。然而，在第一次试验中，男性进入上部隔间时速度稍快，而女性在每次保留试验中接受的电击次数略高。西方饮食（WD）显着损害了雄性苍蝇的学习和记忆，而飞行运动抵消了这种效果。综上所述，苍蝇的被动回避行为提供了一种简单且可重复的测定，可用于研究学习和记忆的基本机制。

引言

学习和记忆是一种进化上古老的环境适应机制，从果蝇（D.）到人类1。果蝇是一种强大的模型生物，用于研究学习和记忆的基本原理，因为它提供了广泛的强大遗传工具来剖析内在的分子机制2。开创性的基因筛查研究确定了对学习和记忆至关重要的rutabaga3，amnesiac4和dunce5基因2，利用了嗅觉调节，因为果蝇依靠其敏锐的嗅觉来寻找食物，潜在的配偶并避开捕食者6。

嗅觉调节已成为研究学习和记忆机制的流行范式，这要归功于Tully和Quinn7，8引入嗅觉T迷宫。随后，已经提出了其他测量各种类型的学习和记忆的方法，包括视觉条件9，求偶条件10，厌恶光速抑制测定11和黄蜂暴露条件反射12。然而，大多数这些检测方法都有一个复杂的设置，必须在大学车间定制或通过供应商购买。这里描述的范式基于一个简单的行为测定，用于研究苍蝇的厌恶联想学习，这些学习可以很容易地与一些可用的用品组装在一起。

所描述的范式相当于实验室小鼠和大鼠的被动（或抑制）回避行为，其中动物学会避开以前接受过电脚电击的隔间13。在Murids中，该程序基于他们对强光的天生回避和对较暗区域的偏好14。在第一次试验中，动物被放入明亮的隔间，从那里动物迅速离开，踏入一个黑暗的隔间，在那里发出电脚电击。通常，一次试验足以形成坚实的长期记忆，导致24小时后的潜伏期显着增加。然后，潜伏期被用作动物记住厌恶刺激与特定环境之间关联的能力的指标15。

这项工作描述了使用 D. 作为模型系统的类似程序，与啮齿动物模型相比，它具有几个优点，包括成本效益，更大的样本量，缺乏监管监督以及获得强大的遗传工具16，17。该过程基于负面的地理出租车行为，这表现在苍蝇被放置在垂直表面上时爬上去的冲动18。该装置由两个垂直腔室组成。在第一次试验中，将果蝇放入较低的隔间。从那里，它通常在3-15秒内离开，进入上层隔间，在那里它受到电击。在1分钟的试验中，一些苍蝇可能偶尔会重新进入上层隔间，从而导致额外的电击。在测试阶段，24小时后，延迟显着增加。同时，与第一天相比，电击次数减少，表明苍蝇在上部隔间形成了厌恶的联想记忆。然后使用延迟，电击次数以及梳理发作的持续时间和频率来分析动物行为以及形成和记住厌恶刺激与特定环境之间关联的能力。具有代表性的结果表明，暴露于西方饮食（WD）会显着损害雄性苍蝇的被动回避行为，这表明WD会深刻影响苍蝇的行为和认知。相反，飞行锻炼减轻了WD的负面影响，改善了被动回避行为。

Access restricted. Please log in or start a trial to view this content.

研究方案

1. 被动回避装置的制备

  1. 钻一个垂直于14 mL聚丙烯培养管壁面的4 mm孔，距离管底部8 mm。
    注:使用电钻和5/32钻头以获得最佳效果。
  2. 使用钢美工刀，切掉14 mL聚丙烯培养管的上部，形成45毫米长的管底部碎片。底部碎片用作下部隔间。
  3. 使用单刃剃须刀片切断1，000 μL蓝色移液器吸头的尖端，使开口足够宽，以便单个苍蝇通过。切掉蓝色尖端的变窄部分，形成一个12毫米的碎片。将此碎片牢固地插入下部隔间的4毫米孔中。这被用作转移苍蝇的装卸月台。
  4. 切割一块 15 mm 的透明乙烯基管，内径为 5/8"（参见 材料表）以创建联轴器。从两端将上部和下部隔间插入联轴器，将下部隔间牢固地连接到上部隔间。
  5. 使用 2 爪可调节夹具，将组件连接到垂直支架上。将组件垂直定向，将减震管作为上部隔间。
  6. 将冲击管导线连接到电刺激器（见 材料表）以提供电击。训练时间为1分钟。
    注意:为了便于观察，请将一张白纸放在减震管后面，作为设备的白色背景。将带有75 W等效软灯泡的灯放在防震室上方。在设置前面放置一个可调节的臂放大灯。被动回避装置的表示 如图1所示。

2. 为被动回避程序准备苍蝇

  1. 使用冰冷块固定3-4天大 的D. melanogaster 或 D. simulans 苍蝇，并在实验前24小时将它们转移到带有食物的单个小瓶中（每瓶1只苍蝇），按照前面描述的程序19。
    注意:这里描述的实验比较了3-4天大的男性 与。雌性苍蝇在 D. melanogaster 和 D. simulans。
  2. 在行为实验之前，对所有小瓶进行编码。为此，为每个组分配一个字母，例如"A"，"B"，"C"等，每个组飞行一个数字。只有在记录和分析了所有数据之后，才显示此代码。每种基因型或治疗方法至少使用20只苍蝇来对抗个体变异。
    注意:执行实验和分析"盲目"允许在评估苍蝇和数据分析的性能时排除偏差。

3. 执行第一次试验

  1. 使用前面描述的飞嘴吸气器（见材料表）20，通过装卸月台将苍蝇从单个小瓶轻轻转移到下部隔间。通过吸气轻轻地将一只苍蝇吸进口中吸气器。通过轻轻吹入装卸月台来存放苍蝇。
    注意:避免在捕捉和装载过程中给动物施加压力。
  2. 在苍蝇被装入下部隔间后，立即启动1分钟计时器和秒表。
    注:秒表用于测量延迟，并用于计数震动次数的计数计数器。
  3. 按下秒表，通过将所有爪子放在网格上，记录苍蝇进入冲击管时的第一个延迟。打开刺激器，对苍蝇进行电击。刺激参数为120伏，持续时间为1000 ms，脉冲/秒（PPS），列车持续时间为2000 ms。
  4. 如果苍蝇重新进入冲击管，则提供额外的冲击。使用计数计数器或基于Arduino的计数器记录1分钟试验期间接受的电击次数（参见 材料表）。如果使用基于Arduino的计数器，请按照以下步骤操作。
    注意:可选的基于Arduino的设备AKM-007（参见 材料表）可用于测量时间，延迟，冲击次数以及通过按下并释放设备上相应按钮来训练每只动物的梳理频率和持续时间。设备上的按钮用于测量延迟，管理和记录电击次数，以及测量梳理比赛的频率和持续时间。
    1. 在步骤 3.2 中 按"开始 "按钮，在步骤 3.3 中按" 震撼 "按钮。
    2. 要记录梳理比赛的持续时间，请在设备上按下 美容 比赛开始时的美容按钮，然后在美容比赛结束时释放此按钮。
      注意:在整个1分钟的试验中测量了梳理发作。广泛的梳理可能表明动物压力21，22。基于Arduino的设备将所有数据作为CSV文件保存到存储卡中。
  5. 在1分钟的试验结束时，轻轻地将苍蝇转移回单个小瓶。记下延迟、收到的冲击次数以及行为中任何显著的变化。
  6. 用70%乙醇清洁下部和冲击室，用不起毛的清洁纸巾擦拭（ 见材料表），然后用吹风机擦干。用下一只苍蝇重复试验。
  7. 行为实验后，用水和无味洗涤剂清洁下部隔间。用70%乙醇擦拭下部隔间和减震室，并风干过夜。

4. 执行第二次试验

  1. 通过在24小时后重复上述程序（步骤3）来执行第二次试验。以与前一天相同的顺序测试苍蝇。

5. 结果分析

  1. 计算试验1和试验2中每组动物的平均潜伏期、平均休克次数和梳理时间。使用 Tukey 检验执行学生 t 检验进行两组比较，或使用 Tukey 检验进行方差分析进行多重比较23。

Access restricted. Please log in or start a trial to view this content.

结果

被动回避在 D. melanogaster （Canton-S）和 D中进行了研究。 模拟。实验比较了连续试验之间的潜伏期和接受冲击的数量。最初，实验是用3-4天大的雄性 D. melanogaster 苍蝇进行的。在24°C的气候受控环境中，在12小时的光暗循环，70%湿度和受控的种群密度下，将苍蝇保持在标准布卢明顿配方饮食中。通过保持所有组的繁殖条件恒定来控制密度。将15只雄性和15只雌性在24°C，70%湿...

Access restricted. Please log in or start a trial to view this content.

讨论

避免威胁性刺激是各种物种从优雅到人类的适应性行为的关键特征32。回避学习程序通常需要逃避厌恶事件，是自1970年代以来常用的行为任务，用于研究实验室啮齿动物的学习和记忆过程133。在主动回避程序中，冷漠的刺激或条件信号（CS）之后是厌恶事件或无条件信号（US），动物通过执行特定的行为任务来学习避免。在被动回避程序...

Access restricted. Please log in or start a trial to view this content.

披露声明

作者声明没有利益冲突。

致谢

这项研究得到了NIH R15ES029673（AKM）的部分支持。

Access restricted. Please log in or start a trial to view this content.

材料

NameCompanyCatalog NumberComments
Bloomington Formulation dietNutri-Fly 66-112Available from Genesee Scientific Inc., San Diego, CA
1000 µL Blue tipFisherNC9546243
17 x 100 mm 14 mL polypropylene culture tubeVWR 60818-689
Aduino-based Automatic Kontrol ModuleIn-houseAKM-007This unit is optional. Complete description, schematics, wiring diagram and a code are provided at the ECU Digital Market - https://digitalmarket.ecu.edu/akmmodule
Dual-Display 2-Channel  Digital Clock/TimerDigi-SenseAO-94440-10https://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
Electronic Finger CounterN/AN/Ahttps://www.amazon.com/gp/product/B01M8IRK6F/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1
Fisherbrand Sparkleen 1 DetergentFisher Scientific04-320-4
Fly mouth aspiratorIn-housePrepared as described in reference 19.
Grass S88 stimulatorN/AN/ACould be replaced with any stimulator which can provide described parameters
Kim-wipesFisher Scientific06-666Kimberly-Clark Professional 34120
Metal block for fly immobilizationIn-house4 x 13 x 23.5cm aluminum block
Nutiva USDA Certified Organic, non-GMO, Red Palm OilNutivaN/Ahttps://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
Shock tubeCelExplorerTMA-201https://www.celexplorer.com/product_detail.asp?id=217&MainType=110&SubType=8
StopwatchAccusplitA601XLNhttps://www.amazon.com/gp/product/B0007ZGZYI/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1
Transparent vinyl tubing (3/4” OD, 5/8” ID)LowesAvaiable from Lowes

参考文献

  1. Kandel, E. R., Dudai, Y., Mayford, M. R. The molecular and systems biology of memory. Cell. 157 (1), 163-186 (2014).
  2. 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).
  3. 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).
  4. Quinn, W. G., Sziber, P. P., Booker, R. The Drosophila memory mutant amnesiac. Nature. 277 (5693), 212-214 (1979).
  5. 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).
  6. Busto, G. U., Cervantes-Sandoval, I., Davis, R. L. Olfactory learning in Drosophila. Physiology. 25 (6), Bethesda, Md. 338-346 (2010).
  7. 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).
  8. 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).
  9. 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).
  10. Koemans, T. S., et al. Drosophila courtship conditioning as a measure of learning and memory. Journal of Visualized Experiments. (124), e55808(2017).
  11. 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).
  12. 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).
  13. Atucha, E., Roozendaal, B. The inhibitory avoidance discrimination task to investigate accuracy of memory. Frontiers in Behavioral Neuroscience. 9, 60(2015).
  14. 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).
  15. 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).
  16. 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).
  17. 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).
  18. 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).
  19. Yang, D. Simple homemade tools to handle fruit flies-Drosophila melanogaster. Journal of Visualized Experiments. (149), e59613(2019).
  20. Barradale, F., Sinha, K., Lebestky, T. Quantification of Drosophila grooming behavior. Journal of Visualized Experiments. (125), e55231(2017).
  21. 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).
  22. Kalueff, A. V., et al. Neurobiology of rodent self-grooming and its value for translational neuroscience. Nature Reviews: Neuroscience. 17 (1), 45-59 (2016).
  23. Motulsky, H. Intuitive biostatistics: A nonmathematical guide to statistical thinking. Fourth edition. , Oxford University Press. (2018).
  24. 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).
  25. Mu, M. D., et al. A limbic circuitry involved in emotional stress-induced grooming. Nature Communications. 11 (1), 2261(2020).
  26. Song, C., Berridge, K. C., Kalueff, A. V. Stressing' rodent self-grooming for neuroscience research. Nature Reviews: Neuroscience. 17 (9), 591(2016).
  27. Wang, C., Chan, J. S., Ren, L., Yan, J. H. Obesity reduces cognitive and motor functions across the lifespan. Neural Plasticity. 2016, 2473081(2016).
  28. 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).
  29. 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).
  30. Lopez-Taboada, I., Gonzalez-Pardo, H., Conejo, N. M. Western Diet: Implications for brain function and behavior. Frontiers in Psychololgy. 11, 564413(2020).
  31. 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).
  32. 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).
  33. Encyclopedia of Neuroscience. Binder, M. D., Hirokawa, N., Windhorst, U. , Springer Berlin Heidelberg. 3093(2009).
  34. 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).
  35. 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).
  36. Ögren, S. O., Stiedl, O. Encyclopedia of Psychopharmacology. Stolerman, I. P. , Springer Berlin Heidelberg. 960-967 (2010).

Access restricted. Please log in or start a trial to view this content.

Erratum


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.

转载和许可

请求许可使用此 JoVE 文章的文本或图形

请求许可

探索更多文章

176

This article has been published

Video Coming Soon

JoVE Logo

政策

使用条款

隐私

联系我们

向图书馆推荐

JoVE 新闻简报

科研

教育

发表

图书馆员

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。