Fear Conditioning Assay in Mouse

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The study of fear memory is important for understanding various anxiety disorders in which patients experience persistent recollections of traumatic events. These memories often involve associations of contextual cues with aversive events; consequently, Pavlovian classical conditioning is commonly used to study contextual fear learning. A form of contextual fear conditioning that is becoming increasingly important as an animal model of anxiety disorders uses predator odor as a fearful stimulus. Innate fear responses to predator odors are well characterized and reliable; however, attempts to use these odors as unconditioned stimuli in fear conditioning paradigms have been highly dependent on experimental setup and have produced inconsistent behavioral results. Here we present a contextual fear conditioning paradigm using coyote urine as the unconditioned stimulus, which has been shown to produce consistent contextual freezing in response to fear learning (Wang et al., 2012).

Materials and Reagents

  1. Mouse C57BL/6, male, 6 weeks to 4 months old (we suggest using mice of a similar age, with ~3 months old being optimal) (Jackson Laboratories)
  2. Paper towel, cut into 2.5 by 2.5 cm squares
  3. Scotch tape
  4. 100% coyote urine (Maine Outdoor Solutions, catalog number: ACOYD )
  5. Ethanol
  6. Cleaning solution (409 and/or Clorox Clean-Up)
  7. Liquid soap


  1. Cylindrical Plexiglas training context 35 cm in diameter and 35 cm tall with a platform base and fitted removable cylindrical wall (custom made by Just Plastics), painted white with 5-6 black distinct visual cues along the wall in basic shapes, each about 10-12 cm in width.
  2. Camera/computer setup to record animal movement – overhead camera with low lighting levels: Bright enough that the computerized tracking system can track the mouse, but dark enough that the mouse is comfortable exploring the environment.
  3. Limelight (Actimetrics) or some other such behavioral tracking system


  1. Handle mouse for 5 min per day for 3 consecutive days prior to beginning the experiment. Handle by holding each mouse and letting it climb around on and explore your hands so that they habituate to the experimenter. Mice can be run successively in groups of 5, but housed individually.
  2. One day before fear conditioning, place the mouse in the training context for 10 min to allow habituation to the context. Fold a piece of tape into a loop and affix to the underside of a paper towel square, taping it to the center of the cylindrical environment. Saturate the paper towel with 20 drops of water before placing the mouse into the context. For contextual consistency, present a paper towel square wetted with water in all sessions except for the predator odor conditioning session. Record all sessions on a computer with Limelight or the behavioral tracking system of your choice.
  3. The next day, expose the mouse to the context for 10 min to take a baseline freezing measure (again placing water in the center of the context). Remove the mouse from the context, and replace the water with a paper towel square saturated with 20 drops of 100% coyote urine. Place the mouse back in the context in the presence of coyote urine for 5 min. Remove the mouse from the contex (as a control, expose other mice to just water in place of the predator odor).
  4. Clean the base of the context thoroughly with 409 and/or Clorox Clean-up and soap several times. Dry and wipe with ethanol (also use ethanol to clean the context between all sessions). Ensure that no coyote odor remains. Meanwhile, air out the conditioning room by opening the door and turning on several fans. If only water has been used, clean the context in the same manner to ensure consistency.
  5. One hour after the conditioning session, give a short-term memory retention test by placing the mouse again in the cleaned context with water for 10 min.
  6. Perform 10 min long-term memory retention tests at 24 h intervals after the conditioning session. This can be repeated for as many days as you wish to continue the study.
  7. Analyze the percentage of time spent freezing for each session. In Limelight, you can export velocity measures for each second and use Excel filters to calculate what percentage of the total time the mouse spent below a certain freezing threshold velocity (0.5 cm/sec or something to that effect).


This protocol is adapted from Wang et al. (2012).


  1. Wang, M. E., Wann, E. G., Yuan, R. K., Ramos Alvarez, M. M., Stead, S. M. and Muzzio, I. A. (2012). Long-term stabilization of place cell remapping produced by a fearful experience. J Neurosci 32(45): 15802-15814.


恐惧记忆的研究对于了解患者经历持续不断创伤性事件的各种焦虑症是重要的。 这些记忆经常涉及到具有厌恶性事件的语境线索的关联; 因此,巴甫洛夫经典条件通常用于研究情境恐惧学习。 作为焦虑症的动物模型正在变得越来越重要的语境恐惧条件的形式使用捕食者气味作为可怕的刺激。 对捕食者气味的先天恐惧反应具有良好的特征和可靠性; 然而,尝试将这些气味用作恐惧调节范例中的无条件刺激已经高度依赖于实验设置,并且已经产生不一致的行为结果。 在这里,我们提出了使用土狼尿液作为无条件刺激的情境恐惧条件范例,已被证明可以产生响应于恐惧学习的一致的语境冷冻(Wang等人,2012)。


  1. 小鼠C57BL/6,雄性,6周至4个月大(我们建议使用具有相似年龄,约3个月大的小鼠是最佳的)(Jackson Laboratories)
  2. 纸巾,切成2.5×2.5厘米的方块
  3. 苏格兰磁带
  4. 100%coyote urine(Maine Outdoor Solutions,目录号:ACOYD)
  5. 乙醇
  6. 清洁溶液(409和/或Clorox清洁)
  7. 肥皂


  1. 圆柱形有机玻璃培训环境直径35厘米,高35厘米,有一个平台基座和装配的可拆卸圆柱形壁(由Just Plastics定制),白色带有5-6黑色不同的视觉提示沿墙壁的基本形状, 宽12cm。
  2. 用于记录动物运动的摄像机/计算机设置 - 具有低照明水平的架空摄像机:足够亮以使得计算机化跟踪系统可以跟踪鼠标,但足够暗以使得鼠标舒适地探索环境。
  3. Limelight(Actimetrics)或某些其他此类行为跟踪系统


  1. 在开始实验前连续3天每天处理小鼠5分钟。抓住每个鼠标,让它爬上来探索你的手,让他们习惯实验者。小鼠可以连续运行,每组5只,但单独饲养。
  2. 前一天恐惧条件,将鼠标在训练上下文中10分钟,以允许习惯上下文。将一条胶带折成一圈,并粘贴到纸巾方格的下侧,将其贴在圆柱形环境的中心。在将鼠标放入上下文之前,用20滴水饱和纸巾。为了上下文一致性,在除了捕食者气味调节期之外,在所有会话中呈现用水润湿的纸巾方格。在使用Limelight或您选择的行为跟踪系统的计算机上记录所有会话。
  3. 第二天,将鼠标暴露在上下文中10分钟,以进行基线冻结测量(再次将水放在上下文的中心)。从上下文中删除鼠标,然后用纸巾替换水 方形饱和20滴100%的土狼尿。在土狼尿存在的情况下放置鼠标5分钟。从contex中删除鼠标(作为控件,暴露其他小鼠只是水代替捕食者的气味)。
  4. 用409和/或Clorox清洁和肥皂清洁上下文几次。干燥并用乙醇擦拭(也用乙醇清洁所有会话之间的上下文)。确保没有土狼气味残留。同时,通过打开门并打开几个风扇的空调室。如果仅使用水,请以相同的方式清洁上下文以确保一致性。
  5. 调节期后一小时,通过将小鼠再次用水清洗10分钟,进行短期记忆保持试验。
  6. 在调节后24小时间隔进行10分钟长期记忆保留试验。这可以重复您想继续学习的天数。
  7. 分析每个会话冻结的时间百分比。在Limelight中,您可以导出每秒的速度测量值,并使用Excel筛选器计算鼠标在某个冻结阈值速度(0.5厘米/秒或其他效果)下所花费的总时间的百分比。




  1. Wang,M.E.,Wann,E.G.,Yuan,R.K.,Ramos Alvarez,M.M.,Stead,S.M.and Muzzio,I.A。(2012)。 由可怕的经验所产生的地点细胞重新对应的长期稳定性。 J Neurosci 32(45):15802-15814。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wang, M. E. and Muzzio, I. A. (2013). Fear Conditioning Assay in Mouse. Bio-protocol 3(7): e531. DOI: 10.21769/BioProtoc.531.
  2. Wang, M. E., Wann, E. G., Yuan, R. K., Ramos Alvarez, M. M., Stead, S. M. and Muzzio, I. A. (2012). Long-term stabilization of place cell remapping produced by a fearful experience. J Neurosci 32(45): 15802-15814.

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