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。
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。
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。
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
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1. 被动回避装置的准备
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被动回避实验在 D. melanogaster （中国台湾） 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。果蝇饲养于标准Bloomington配方培养基上，在温度24 °C、12小时光照-黑暗循环、70%湿度及控制种群密度的恒温环境条件下进行培养。通过保持所有组别的繁殖条件一致来控制种群密度。每瓶中放置15只雄蝇和15只雌蝇，在24 °C、70%湿度及12小时光照周期条件下交配48小时以产生子代。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。所有实验均在每天相同时间点进行。实验过程中，将单个离心管中的果蝇轻轻吸出，并转移至下层隔室 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，并常会重复进入，平均受到2次电击（2.18.......
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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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作者声明无利益冲突。
本研究部分由美国国立卫生研究院（NIH）R15ES029673（AKM）资助。
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| 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 |
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申请许可以重复使用本 JoVE 文章的文本或图表
申请许可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.