Two-choice Digging Task in Mouse for Studying the Cognitive Flexibility

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Nature Neuroscience
Jan 2014



Cognitive flexibility, the higher-order cognition involving reversal learning, has been defined as having the ability to shift one’s previous thoughts or actions to new situations depending on situational demands. Studies of neuropsychiatric disorders such as autism spectrum disorder (ASD) showed that restricted and repetitive patterns of activities are associated with the impairments of cognitive flexibility. Some behavioral tasks including attentional set-shifting task are used to assess cognitive flexibility in mouse models for psychiatric disorders (Birrell and Brown, 2000; Colacicco et al., 2002). Here we present a two-choice digging test, which is simplified and modified from set-shifting task, for using mice to study the reversal learning (Huang et al., 2014).

Keywords: Mouse behavior (老鼠行为), Cognitive flexibility (认知灵活性), Rigidity (硬度)

Materials and Reagents

  1. C57BL/6 mice (purchased from National Laboratory Animal Center)
  2. Sterilized sunflower seeds (SunOpta, catalog number: 10AF4167 )
  3. Cinnamon (Local bakery shop)
  4. Sawdust (P.J. Murphy Forest Products Corporation, Sani-Chips®)
  5. 70% ethanol


  1. Apparatus
    The apparatus (Figure 1) is made of opaque acrylic with a platform base and wall with two transparent plexiglas guillotine doors (25 x 20 x 15 cm). A waiting compartment (20 x 10 cm) at one end of the apparatus connected to two equally sized choice compartments at the other end via guillotine doors (15 x 10 cm).

    Figure 1. The apparatus of two-choice digging task

  2. Digging bowl
    The digging bowl (45 mm in diameter, 25 mm in height) placed in each choice compartment is baited with sunflower seeds (30-35 mg) hidden underneath original- or cinnamon (2%)-flavored sawdust.
    Note: The rewards should be buried under over 20 mm of sawdust.

    Figure 2. The digging bowl


The GraphPad Prism 5.0 (GraphPad Software) was used to generate the graphs and analyze the data of trials to acquisition and trials to relearn. A p-value less than 0.05 is considered statistically significant.



  1. The procedure used in this task is modified from the procedure described for investigating the attentional set-shifting in mice (Colacicco et al., 2002, Garner et al., 2006).
  2. In general, this two-choice digging test consists of a 3-day habituation period, 1-day learning period, and 1-day reversal learning period.
  3. Before starting the task and then throughout the entire experiment, mice are housed individually in their home cages and are food-restricted (1.5-2 g per day) to reduce their body weight to 85-90% of the free-feeding body weight.

    1. 3-day habituation period
      In order to ensure that the subject mice dig for rewards of sunflower seeds in digging bowls placed in each reward chamber, the mice must be habituated to the baited bowls filled with original- or cinnamon-flavored sawdust. To do this, mice are deprived of food for 22 h and then allowed to daily access to wet mashed food for 2 h every day. Following feeding, a digging bowl filled with sawdust and baited with sunflower seeds is left in their home cage overnight, so that mice could be trained to habituate the experimental tools and learn to retrieve the reward by digging in the bowl. The following steps are taken for this period.
      1. Day 1
        1. After the 22-h food deprivation, feed the mouse daily mashed and moisture food (1.5-2 g) for 2 h.
        2. After mice consumed moisture food, place a digging bowl baited with sunflower seeds (30-35 mg) hidden underneath original-flavored sawdust into home cage overnight and resume the 22-h food deprivation.
      2. Day 2 (Video 1)
        1. Mice are placed into the waiting compartment.
        2. Once the mouse is not facing or touching a door, both guillotine doors are lifted simultaneously to allow the mouse to explore the test box freely for 10 min.
        3. After 10 min has expired, return mouse back to the waiting chamber and lower the doors immediately. Meanwhile, place an original sawdust-filled baited bowl in each choice compartment simultaneously.
        4. Reopen both doors, mice are then given two consecutive trials with unlimited free access to both baited bowls in the testing apparatus until both rewards are found. Once mice retrieve a reward, they are allowed to consume it before being returned to the waiting compartment.
        5. As the trials are complete, return mice to their home cages. Feed mice with the wet mashed food (1.5-2 g) for 2 h.
        6. After 2 h has elapsed, place a 2% cinnamon-flavored sawdust-filled baited bowl into the home cages overnight and resume the 22-h food deprivation.
          Note: The 2% cinnamon-flavored sawdust could be prepared by evenly mixing the sawdust and cinnamon in the ziplock bag. During the preparation, change gloves after weighting out or touching cinnamon to avoid the cross contamination between original- and cinnamon-flavored sawdust.

          Video 1. Two-choice digging-Day 2-Habituation period

      3. Day 3: Almost the same as day 2 except that the original sawdust-filled baited bowls are replaced by 2% cinnamon-flavored sawdust-filled baited bowls in the choice chambers.
        1. After the 22-h food deprivation, place mice into the waiting compartment, followed by 10 min of free exploration for the test box.
        2. Immediately after exploration, mice are given two consecutive trials to freely access two 2% cinnamon-flavored sawdust-filled baited bowls positioned in choice compartments until both rewards are retrieved and consumed.
        3. Return mice to home cage and feed them with restricted food. After one hour feeding, place original sawdust-filled baited bowls into their cage overnight and resume the food deprivation. After three days of habituation, mice should be trained to accustom to the task structure and find food rewards by digging in the food bowls rapidly.

    2. 1-day learning period
      In this period, mice are trained to learn that the bowl filled with original-flavored sawdust is baited (correct) and 2% cinnamon-flavored sawdust is unbaited (incorrect). This learning test consists of training trials (first four trials) and testing trials (from trial 5 onwards).
      1. Day 4 (Video 2)

        Video 2. Two-choice digging-Day 4-learning period

        1. Place the digging bowls in choice compartments: One is original sawdust-filled baited; the other is cinnamon-flavored sawdust-filled unbaited.
          Note: The baited and unbaited bowls are positioned randomly in each of choice compartments (left versus right). For each mouse, the baited bowl should be presented at the left and right hands simultaneously to prevent spatially cueing the mouse as to which bowl is baited. Throughout this period, baited bowls should not be presented in the same choice compartment more than three times in a row.
        2. After food deprivation, place mice in the waiting compartment and then open the guillotine doors simultaneously to begin the training trials.
        3. In the training trials, mice are received first four trials, in which they are allowed to freely dig in both bowls. If the first dig occurred in the baited bowl, mice are allowed to retrieve and consume the rewards and then dig in the other unbaited bowl. If the first dig occurred in the unbaited bowl, an incorrect choice is recorded and mice are still allowed to retrieve the reward from the other baited bowl.
          Note: Only moving the sawdust from bowls with paws or nose can be defined as the digging response. The simply contacting or sniffing the sawdust or bowl performed by mice can’t be recognized as the digging choice.
        4. After one training trial is complete, mice are returned to the waiting compartment and place to their home cage waiting for the next trial. Meanwhile, clean the base, wall, and doors of the apparatus thoroughly with ethanol to ensure that no odor remains (ethanol should to be used to clean this apparatus carefully between all trials).
          Note: No more than four mice are tested as a group in this task to avoid waiting too long for the next trial.
        5. After four training trials are complete, mice are subjected to testing trials from trial 5 onwards, in which mice are allowed to dig in one bowl only. If mice first dig in the original odor sawdust-filled baited bowl (correct), they are allowed to consume the reward. The trial is terminated and the mouse is returned to the waiting compartment after digging when the mouse starts to dig in the cinnamon odor sawdust-filled unbaited bowl (incorrect). Once one testing trial is complete, the mouse will be returned to his cage, waiting for the next trial.
          Note: If no choice is made 5 min after trial beginning, the trial is terminated and marked as incomplete.
        6. The bowl that the mouse chooses to first dig in (correct/incorrect) is recorded. The testing trial is repeated for many times. Once the mouse meets a criterion level of six consecutive correct trials, the testing is ended. The number of trials needed to reach the criterion is recorded.
        7. After the testing trials is complete, return the mouse to its home cage and give mouse the restricted chow pellets (1.5-2 g) overnight.

    3. 1-day reversal learning period
      On the next day, mice are subjected to the reversal learning period, in which the reward is switched to the bowl filled with 2% cinnamon-flavored sawdust. Mice are again first given training trials (first four trials) to relearn that the cinnamon odor sawdust-filled bowl is the correct one and then allowed to immediately choose one bowl only to test their cognitive flexibility in the testing trials (from trial 5 onwards).
      1. Day 5: Same as day 4 (the learning period) except that the reward is buried under the 2% cinnamon odor sawdust, meaning the bowl filled with 2% cinnamon-flavored sawdust is baited (correct) and the bowl filled with original-flavored sawdust is unbaited (incorrect).
        1. Place the bowls, one is cinnamon odor (baited/correct) and the other is original odor (unbaited/incorrect), in the choice compartments.
        2. In the first four trials (training trials), mice are allowed to dig in both baited and unbaited bowls.
        3. After training, the testing trials are begun from trial 5 onwards, in which mice are allowed to dig in one bowl only.
        4. Testing is terminated once mouse reaches the criterion of six consecutive correct trials. Record the number of trials needed to meet the criterion.


  1. Flavored sawdust
    2% cinnamon


This protocol was adapted from previous works listed in the references.


  1. Birrell, J. M. and Brown, V. J. (2000). Medial frontal cortex mediates perceptual attentional set shifting in the rat. J Neurosci 20(11): 4320-4324.
  2. Colacicco, G., Welzl, H., Lipp, H. P. and Wurbel, H. (2002). Attentional set-shifting in mice: modification of a rat paradigm, and evidence for strain-dependent variation. Behav Brain Res 132(1): 95-102.
  3. Garner, J. P., Thogerson, C. M., Würbel, H., Murray, J. D. and Mench, J. A. (2006). Animal neuropsychology: validation of the Intra-Dimensional Extra-Dimensional set shifting task for mice. Behav Brain Res 173(1): 53-61.
  4. Huang, T. N., Chuang, H. C., Chou, W. H., Chen, C. Y., Wang, H. F., Chou, S. J. and Hsueh, Y. P. (2014). Tbr1 haploinsufficiency impairs amygdalar axonal projections and results in cognitive abnormality. Nat Neurosci 17(2): 240-247.


认知灵活性,涉及反向学习的高阶认知,已被定义为具有根据情境需求将一个先前的想法或行动转移到新的情况的能力。 神经精神疾病如自闭症谱系障碍(ASD)的研究表明,受限和重复的活动模式与认知灵活性的损伤有关。 包括注意集移位任务的一些行为任务用于评估用于精神障碍的小鼠模型中的认知灵活性(Birrell和Brown,2000; Colacicco等人,2002)。 在这里,我们提出了一个双选择挖掘测试,这是简化和修改从集移位任务,使用小鼠来研究逆转学习(黄等人。,2014)。

关键字:老鼠行为, 认知灵活性, 硬度


  1. C57BL/6小鼠(购自National Laboratory Animal Center)
  2. 灭菌的向日葵种子(SunOpta,目录号:10AF4167)
  3. 肉桂(当地面包店)
  4. 锯屑(P.J.Murphy Forest Products Corporation,Sani-Chips )
  5. 70%乙醇


  1. 装置
    该设备(图1)由具有平台基部和具有两个透明有机玻璃闸门(25×20×15cm)的不透明丙烯酸制成。 设备一端的等待舱(20×10厘米)通过闸门(15×10厘米)连接到另一端的两个相同大小的选择隔间。


  2. 挖坑
    将置于每个选择隔室中的挖碗(直径45mm,高25mm)用隐藏在原始或肉桂(2%)风味锯木下的向日葵种子(30-35mg)饵料化。 注意:奖励应埋在超过20毫米的锯末上。



GraphPad Prism 5.0(GraphPad软件)用于生成图表并分析试验的数据以获取和重新学习试验。 小于0.05的p值被认为是统计学显着的。



  1. 该任务中使用的程序从用于研究小鼠的注意力集中转移的程序(Colacicco等人,2002,Garner等人,2006)修改。
  2. 一般来说,这种双选择挖掘测试包括3天适应期,1天学习期和1天反转学习期。
  3. 在开始任务之前,然后在整个实验中,将小鼠单独圈养在它们的家笼中,并且限制食物(每天1.5-2g),以将其体重降低至自由进食的85-90%体重。

    1. 3天适应期
      1. 第1天
        1. 在22小时食物剥夺之后,每天喂食小鼠捣碎和水分食物(1.5-2g)2小时。
        2. 小鼠消耗水分食物后,放置一个挖坑碗诱饵 向日葵种子(30-35毫克)隐藏在原始风味的锯木屑下面 进入家笼,并恢复22小时的食物剥夺。
      2. 第2天(视频1)
        1. 小鼠被放入等候室。
        2. 一旦鼠标没有面对或触摸门,两个断头台 门同时提起,以允许鼠标探索测试 盒子10分钟。
        3. 10分钟后过期,返回鼠标 返回到等待室并立即降低门。 与此同时, 在每个选择的隔间放置一个原始锯木屑填充的诱饵碗 同时。
        4. 重新打开两门,小鼠然后给两个 连续试验与无限自由访问两个诱饵碗 测试装置直到找到两个奖励。 一旦老鼠检索a   奖励,他们被允许消费它,然后才返回 等候室。
        5. 当试验完成后,将小鼠送回他们的家笼。 饲料小鼠与湿捣碎食品(1.5-2克)2小时。
        6. 2小时后,放入2%肉桂风味的锯屑 诱饵碗进入家笼子过夜,恢复22小时的食物 剥夺。
          注意:可以制备2%肉桂味的锯屑  通过均匀地混合锯末和肉桂在ziplock袋。中 准备,改变手套后加重或接触肉桂 以避免原始和肉桂风味之间的交叉污染  锯末。

          视频1.两选择挖掘 - 第2天 - 习惯时期
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      3. 第3天:几乎和第2天相同,只是原来的 锯末填充的诱饵碗由2%肉桂风味代替 锯末填充的诱饵碗在选择室。
        1. 之后 22小时食物剥夺,将小鼠放入等候室,然后   通过10分钟的自由探索测试箱
        2. 立即 在探索之后,小鼠被自由地连续两次试验 访问两个2%肉桂风味的锯屑填充的诱饵碗定位 在选择隔间,直到两个奖励被检索和消耗
        3. 将小鼠回到笼子里,喂食限制食物。 后 一小时喂食,将原来锯木屑填充的诱饵碗放入他们的 笼子过夜,恢复食物剥夺。 三天后 习惯,小鼠应该训练习惯于任务结构 并通过在食物碗中快速找到食物的奖励。

    2. 1天学习期
      在这个时期,小鼠训练得知,装满原始味的锯木屑的碗被诱饵(正确)和2%肉桂风味锯木屑是不挨打的(不正确)。 这种学习测试包括训练测试(前四个测试)和测试试验(从试验5开始)。
      1. 第4天(视频2)

        视频2.双向选择挖掘 - 第4天学习期
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        1. 将挖碗放在选择的隔间:一个是原始的 锯末填充的诱饵; 另一种是肉桂风味的锯末填充 不羁。
          注意:诱饵和不ited碗位于 在每个选择的区室(左对右)随机。 每个 鼠标,诱饵碗应该呈现在左右手 同时防止空间提示鼠标关于哪个碗是   诱饵。 在整个这段时间,诱饵碗不应该被提出   相同的选择室连续三次以上。
        2. 食物剥夺后,将小鼠放在等候室,然后 同时打开闸门开始训练试验
        3. 在训练试验中,小鼠接受前四个试验,其中   他们被允许自由地在两个碗里挖。 如果第一次发生   在诱饵碗中,允许小鼠检索和消费 奖励,然后挖在另一个unbaited碗。 如果第一次挖 发生在未被褥的碗,记录不正确的选择和老鼠 仍然允许从另一个诱饵碗中取回奖励 注意:   只有用爪子或鼻子从碗中移动锯屑可以定义为 挖掘响应。 简单地接触或嗅探锯末或 由小鼠执行的碗不能被识别为挖掘选择。
        4. 一个训练试验完成后,小鼠回到 等待隔间和地方到他们的家庭笼子等待下 试验。同时,清洁设备的基座,墙壁和门 彻底用乙醇,以确保没有气味残留(乙醇应该 用于在所有试验之间仔细清洁此设备) 注意:在此任务中,测试不超过四只小鼠作为一组,以避免下次试验等待时间过长。
        5. 四个训练试验完成后,对小鼠进行 从试验5起的测试试验,其中允许小鼠挖入  一碗只。如果老鼠先挖了原来的气味锯屑填充 诱饵碗(正确),他们被允许消耗奖励。试用  并且鼠标返回到等待室 后挖掘时,老鼠开始挖肉桂的气味 锯末填充的不褥碗(不正确)。一次测试试验 完成后,鼠标会回到他的笼子里,等待下一个 试验。
        6. 鼠标选择首先挖的碗(正确/不正确) 。 测试试验重复多次。 一旦 小鼠符合六个连续正确试验的标准水平 测试结束。 达到标准所需的试验次数是 已记录。
        7. 测试试验完成后,返回 小鼠到其家笼,并给小鼠限制性食物颗粒(1.5-2   g)过夜。

    3. 1天反转学习期
      第二天,将小鼠进行逆转学习期,其中将奖励切换到填充有2%肉桂风味锯木屑的碗中。 小鼠再次首先进行训练试验(前四个试验)以重新学习 肉桂气味锯屑填充碗是正确的,然后允许立即选择一个碗只是为了测试他们的认知灵活性在测试试验(从试验5起)。
      1. 第5天:与第4天(学习期)相同,除了奖励是 掩埋在2%肉桂味锯屑下,意味着碗里充满了 2%肉桂风味的锯屑被诱饵(正确)和碗填充 与原始风味的锯木屑是不羁的(不正确)。
        1. 地点  碗,一个是肉桂味(诱饵/正确),另一个是 原始气味(未ited /不正确),在选择隔间
        2. 在前四个试验(训练试验)中,允许小鼠在诱饵和非诱饵碗中进行挖掘
        3. 训练后,从试验5开始测试试验,其中允许小鼠仅在一个碗中挖。
        4. 一旦小鼠达到6的标准,测试终止 连续正确试验。 记录需要满足的试验次数 标准。


  1. 调味锯末




  1. Birrell,J.M。和Brown,V.J。(2000)。 内侧额叶皮质介导大鼠的知觉注意集移位。 J Neurosci 20(11):4320-4324
  2. Colacicco,G.,Welzl,H.,Lipp,H.P.and Wurbel,H。(2002)。 小鼠的注意力转移:大鼠范例的修改,以及应变依赖性变异的证据。 Behav Brain Res 132(1):95-102。
  3. Garner,J.P.,Thogerson,C.M.,Würbel,H.,Murray,J.D.and Mench,J.A。(2006)。 动物神经心理学:验证小鼠的内部维度外形集移位任务。 Behav Brain Res 173(1):53-61。
  4. Huang,T.N.,Chuang,H.C.,Chou,W.H.,Chen,C.Y.,Wang,H.F.,Chou,S.J.and Hsueh,Y.P.(2014)。 Tbr1 haploinsufficiency损害杏仁核轴突预测和结果的认知异常。 Nat Neurosci 17(2):240-247。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chuang, H., Huang, T. and Hsueh, Y. (2014). Two-choice Digging Task in Mouse for Studying the Cognitive Flexibility. Bio-protocol 4(19): e1250. DOI: 10.21769/BioProtoc.1250.



Labeeba Nusrat
University of Toronto

i was wondering how much Cinnamon to use. in a large ziplog bag if i made flavoured sawdust, how do i know how much is 2%? Do i measure the sawdust amount and calculate 2% with respect to that?

Hope to hear back from you soon. Thanks,

Labeeba Nusrat, MSc. Candidate
Morshead Lab
University of Toronto
4/19/2015 6:22:48 AM Reply
Yi-Ping Hsueh
Institute of Molecular Biology, Academia Sinica, Taiwan

Hi Labeeba,

The 2% cinnamon-flavored sawdust is calculated as the fraction of the weight of cinnamon related to the total weight of the cinnamon-flavored sawdust (weight percentage, w/w). I mix ground cinnamon with sawdust to give a 2% (w/w) cinnamon mixture. For example, if I want to prepare 100g 2% cinnamon-flavored sawdust, I measure 2g cinnamon amount and mix the 2g cinnamon with sawdust (~98g) to reach 100g of cinnamon-sawdust mixture. I calculate the weight of sawdust or cinnamon by weighing equipment and put/mix them in a large ziplog bag. I hope these information will be helpful. If you have more questions, I will be happy to provide any information.

4/19/2015 10:54:22 PM