Olfactory Avoidance Test (Mouse)
嗅觉回避试验(小鼠)   

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本实验方案简略版
The Journal of Neuroscience
Aug 2016

 

Abstract

In mice, olfaction plays a pivotal role for the various behaviors, such as feeding, mating, nursing and avoidance. Behavioral tests that analyze abilities of odor detection and recognition using genetically modified mice reveal the contribution of target genes to the olfactory processing. Here, we describe the olfactory avoidance test to investigate the odor detection ability in mice.

Keywords: Olfaction (嗅觉), Odor detection threshold (气味检测阈值), Behavioral test (行为测试), Mouse (小鼠), Freezing (冷冻), Avoidance (回避)

Background

Olfactory system is a good model for studying the sensory processing in the brain. To characterize innate fear responses such as freezing and avoidance in genetically modified mice, the olfactory avoidance test was performed using a component of fox feces, TMT (2,5-dihydro-2,4,5-trimethylthiazoline; Kobayakawa et al., 2007). Furthermore, the olfactory avoidance using the different amounts of TMT was carried out to know the odor detection threshold in gene-knockout mice (Kaneko-Goto et al., 2013). Recently, we have reported that non-dihydrogenated TMT (nTMT: 2,4,5-trimethylthiazole) also induces similar freezing and avoidance responses (Takahashi et al., 2016). Here, we describe a method for the olfactory avoidance test with nTMT (commercially available) to explore the odor detection threshold in mice. This method has an advantage in the point using a simple device such as cage and filter paper, compare with that using an olfactometer.

Materials and Reagents

  1. Latex gloves (NIPPON Genetics, catalog number: SLPF-M )
  2. Filter paper (3 MM CHR) (GE Healthcare, catalog number: 3030-909 )
  3. Paper towels (KCWW, Kimberly-Clark, catalog number: 47000 )
  4. Laboratory-bred mice
    Note: For the test, male mice should be used to avoid the effect of the estrous cycle. Mice are housed in groups 3-5 per cage, kept in a room with controlled temperature (~23 °C) and humidity under 12 h light/dark cycle (lights on at 8:30 AM) with ad libitum access to food and water.
  5. 2,4,5-trimethylthiazole (nTMT) (7.9 M) (Tokyo Chemical Industry, catalog number: T1068 )
    Note: A component of fox feces, TMT (2,5-dihydro-2,4,5-trimethylthiazoline), is known to evoke innate fear responses in rodents (Kobayakawa et al., 2007; Kaneko-Goto et al., 2013). Non-dihydrogenated TMT (nTMT) also induces similar freezing and avoidance responses (Takahashi et al., 2016).
  6. 70% ethanol

Equipment

  1. Test cage (31 x 21 x 12.5 cm) (one cage per one mouse)
  2. Habituation cage with the same size as the test cage (four cages per one mouse)
  3. Clear acrylic board (which can cover the roof of the test cage)
  4. Video camera (Sony, catalog number: HDR-CX560V )
    Note: The mouse behavior is recorded under the weak-light condition. This model (Sony Nightshot Camcorder) is equipped with infrared-mode.
  5. Tripod for camera (SLIK, catalog number: F 740 )
  6. Red light
  7. Fume hood

Software

  1. Microsoft Excel (Microsoft)

Procedure

  1. Before the test day
    1. During five days before the test, mice are habituated to the experimental condition, under which an experimenter handles them with latex gloves on. The same experimenter gently handles each mouse for 10 min per day during the five consecutive days.

  2. On the test day (Figure 1)
    1. Tests are done during the dark phase of 12 h light/dark cycle in the test room, in which, the weak red light (< 5 lux) is used for indirect lighting. Individual mice are transferred from their home cage to each habituation cage in the habituation room. The mice are habituated to the experimental environment in the habituation cage for 30 min (Habituation phase). We recommend that the test is performed in the dedicated room, which is different from the habituation room, because it is important to prevent the mice from encountering nTMT before the test.
    2. Each mouse is transferred from an old to a new habituation cage. This habituation process is repeated four times for each animal (use four habituation cages per one mouse, totally for 2 h) (Habituation phase; Figure 1A, top).
    3. Set the test cage and a digital video camera in the test room. Put on a filter paper (2 x 2 cm) at the one side of cage. Cover the roof of the test cage with acrylic board to prevent the odor from diffusing and the mouse from escaping from the test cage (Figure 1B).
    4. Undiluted nTMT at the amount of 0, 0.4, 4, and 40 μl is dropped on the filter paper in the test cage.
    5. After the mouse is transferred to the test cage, record the behavior with a video camera during the 10-min test (Test phase; Figure 1A, bottom).
    6. The tested mouse is returned to their home cage. After the one test finishes, the used cage and filter paper scented with nTMT should be kept in the fume hood (see Note 4). Start the next test using new test cage and another mouse.
    7. The recorded videos are analyzed, as described below.


      Figure 1. Olfactory avoidance test. A. Schema of the experimental procedure; B. Apparatus of the olfactory avoidance test.

Data analysis

Both freezing and avoidance times are measured manually during the 10-min test using the recorded videos. ‘Freezing time’ is defined as the time kept still for more than 3 sec, except for breathing (Figure 2A and Video 1). ‘Avoidance time’ is defined as the time spent in an area without a filter paper scented with nTMT, when the test cage is divided into two equal areas. Avoidance behavior is represented by an avoidance index (avoidance index = [P - 50]/50, where P is the percentage of avoidance time during the 10-min test period; Figure 2B). We recommend that both freezing and avoidance times are measured by blinded analysis.
P-values are calculated by Welch t-test using Microsoft Excel, in which you click Data Analysis and perform t-test: Two-Sample Assuming Unequal Variances. For multiple pairwise comparisons, P-values are then sequentially evaluated according to the Holm-Bonferroni method (Holm, 1979) to keep an experiment-wise α ≤ 0.05, manually.
The formula to evaluate the Holm-Bonferroni method is as follows:
α/(n - k + 1)
Where,
n: number of tests,
k: rank number of pair.
Example for the Holm-Bonferroni correction:
Consider four null hypotheses (H1-4) with unadjusted P-values (p1-4), to be tested at significance level α = 0.05. H1: p1 = 0.01, H2: p2 = 0.003, H3: p3 = 0.03, H4: p4 = 0.04

  1. Order the P-values from smallest to largest.
    p2 (= 0.003) < p1 (= 0.01) < p3 (= 0.03) < p4 (= 0.04)
  2. To calculate the adjusted alpha level, work the Holm-Bonferroni formula for the first rank, and compare it with the first-ranked P-value (p2). If the P-value is smaller, reject the first-ranked null hypothesis (H2).
    α/(n - k + 1) = 0.05/(4 - 1 + 1) = 0.0125
    0.0125 > p2 (= 0.003). H2 is rejected.
  3. Repeat the Holm-Bonferroni formula for the second rank, and compare it with the second-ranked P-value (p1).
    α/(n - k + 1) = 0.05/(4 - 2 + 1) = 0.0167
    0.0167 > p1 (= 0.01). H1 is rejected.
  4. Repeat the Holm-Bonferroni formula for the third rank, and compare it with the third-ranked P-value (p3).
    α/(n - k + 1) = 0.05/(4 - 3 + 1) = 0.0250
    0.0250 < p3 (= 0.03). H3 is not rejected.
  5. The testing stops when you reach the first non-rejected hypothesis. All subsequent hypotheses are non-significant. We conclude that H1 and H2 are rejected and H3 and H4 are not rejected.


    Figure 2. Sample data of the olfactory avoidance test. A and B. Freezing time (A) and avoidance index (B) in wild-type mice; C. When a lower amount (4 μl) of nTMT was employed, 5T4 knockout mice remarkably reduced the freezing time, compared with wild-type mice (see the detail in Takahashi et al., 2016).

    Video 1. Freezing response in wild-type mice for 40 μl nTMT

Notes

  1. For this test, mice should be used only once to avoid confounding of data, based on the learning and memory.
  2. The experimenter must restrict from wearing odorant products with strong smell and from making any excessive noise during the test. Because it is difficult to completely remove the personal smell from the experimenter, we recommend that the same experimenter, who handles the mice for the habituation before the test, performs all the processes throughout the test.
  3. It is important to prevent the odor diffusion in the test room, because the undiluted odor is used in this test. The bottle of nTMT and their derivatives should be preserved in the fume hood. After the one test finishes, the used cage and filter paper scented with nTMT should be kept in the fume hood. In the test, a smaller amount of nTMT should be used first, compared with the larger amount. We recommend that the test is performed in the dedicated room, which is different from the habituation room, because it is important to prevent the mice from encountering nTMT before the test.
  4. The existence of experimenter affects the mouse behaviors in a significant way. The experimenter must depart enough from the test cage. We recommend that when the test phase starts in the dedicated test room, the experimenter gets out there.

Acknowledgments

This protocol was adopted from previous studies (Kobayakawa et al., 2007; Kaneko-Goto et al., 2013). This work was supported by Grants-in-Aid for Scientific Research on (B) (A.T.), (C) (H.T.), and Innovative Areas (Adaptive circuit shift) (A.T.), and for Challenging Exploratory Research (A.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

References

  1. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scand J Statist 6(2): 65-70.
  2. Kaneko-Goto, T., Sato, Y., Katada, S., Kinameri, E., Yoshihara, S., Nishiyori, A., Kimura, M., Fujita, H., Touhara, K., Reed, R. R. and Yoshihara, Y. (2013). Goofy coordinates the acuity of olfactory signaling. J Neurosci 33(32): 12987-12996.
  3. Kobayakawa, K., Kobayakawa, R., Matsumoto, H., Oka, Y., Imai, T., Ikawa, M., Okabe, M., Ikeda, T., Itohara, S., Kikusui, T., Mori, K. and Sakano, H. (2007). Innate versus learned odour processing in the mouse olfactory bulb. Nature 450(7169): 503-508.
  4. Takahashi, H., Ogawa, Y., Yoshihara, S., Asahina, R., Kinoshita, M., Kitano, T., Kitsuki, M., Tatsumi, K., Okuda, M., Tatsumi, K., Wanaka, A., Hirai, H., Stern, P. L. and Tsuboi, A. (2016). A subtype of olfactory bulb interneurons is required for odor detection and discrimination behaviors. J Neurosci 36(31): 8210-8227.

简介

在小鼠中,嗅觉对于各种行为,如喂养,交配,护理和避孕都起着关键的作用。 使用基因修饰小鼠分析气味检测和识别能力的行为测试揭示了目标基因对嗅觉加工的贡献。 在这里,我们描述了嗅觉回避试验,以调查小鼠的气味检测能力。
【背景】嗅觉系统是研究大脑感觉处理的好模型。 为了表征在转基因小鼠中的先天性恐惧反应如冷冻和避免,使用狐狸粪便组分TMT(2,5-二氢-2,4,5-三甲基噻唑啉; Kobayakawa等,2007)进行嗅觉回避试验)。 此外,进行使用不同量的TMT的嗅觉避免以了解基因敲除小鼠的气味检测阈值(Kaneko-Goto等,2013)。 最近,我们已经报道非脱氢TMT(nTMT:2,4,5-三甲基噻唑)也诱导类似的冷冻和回避反应(Takahashi等,2016)。 在这里,我们描述了使用nTMT(市售)进行嗅觉回避试验以探索小鼠气味检测阈值的方法。 该方法在使用诸如笼和滤纸的简单装置方面具有优点,与使用嗅觉计的那些相比。

关键字:嗅觉, 气味检测阈值, 行为测试, 小鼠, 冷冻, 回避

材料和试剂

  1. 乳胶手套(NIPPON Genetics,目录号:SLPF-M)
  2. 滤纸(3 MM CHR)(GE Healthcare,目录号:3030-909)
  3. 纸巾(KCWW,Kimberly-Clark,目录号:47000)
  4. 实验室繁殖小鼠
    注意:对于测试,应使用雄性小鼠避免发情周期的影响。小鼠以每笼3-5个饲养,保存在受控温度(〜23℃)和12小时光/黑暗循环(8:30 AM点亮)下的湿度,随意获取食物和水
  5. 2,4,5-三甲基噻唑(nTMT)(7.9M)(Tokyo Chemical Industry,目录号:T1068)
    注意:已知狐狸粪便组分TMT(2,5-二氢-2,4,5-三甲基噻唑啉)在啮齿动物中引起天生的恐惧反应(Kobayakawa et al。,2007; Kaneko-Goto et al。 2013年)。非脱氢TMT(nTMT)也诱导类似的冷冻和回避反应(Takahashi等,2016)。
  6. 70%乙醇

设备

  1. 测试笼(31 x 21 x 12.5 cm)(每个鼠标一个笼子)
  2. 具有与测试笼相同尺寸的习惯笼(每个鼠标四个笼)
  3. 透明丙烯酸板(可覆盖测试笼的屋顶)
  4. 摄像机(Sony,目录号:HDR-CX560V)
    注意:在弱光条件下记录鼠标行为。该型号(Sony Nightshot摄像机)配备了红外模式。
  5. 三脚架相机(SLIK,目录号:F 740)
  6. 红灯
  7. 通风柜

软件

  1. Microsoft Excel(Microsoft)

程序

  1. 考试日之前
    1. 在测试前五天,小鼠习惯于实验条件,实验者用乳胶手套处理它们。相同的实验者在连续五天内每天轻轻地处理每只小鼠10分钟。

  2. 在测试日(图1)
    1. 在测试室中的12小时光/暗循环的暗相期间进行测试,其中弱红光(<5勒克斯)用于间接照明。将个体小鼠从他们的家笼转移到习惯室中的每个习惯笼。小鼠在习惯性笼中习惯于实验环境30分钟(习惯阶段)。我们建议在专门的室内进行测试,这是不同于习惯的房间,因为防止老鼠在测试前遇到nTMT是很重要的。
    2. 每只老鼠从一个旧的笼子转移到一个新的习惯。对于每只动物,这种习惯过程重复四次(每个小鼠使用四个习惯笼,总共2小时)(习惯阶段;图1A,上)。
    3. 在测试室中设置测试笼和数码摄像机。在笼子的一侧放置一张滤纸(2 x 2厘米)。用丙烯酸板覆盖测试笼的屋顶,以防止气味扩散,鼠标从测试笼中逸出(图1B)。
    4. 在测试笼中的滤纸上滴下0,0.4,4和40μl量的未稀释的nTMT。
    5. 将鼠标转移到测试笼后,在10分钟测试(测试阶段;图1A,底部)中记录摄像机的行为。
    6. 被测试的鼠标返回到他们的家笼。一次测试结束后,使用nTMT香味的笼子和滤纸应保存在通风橱中(见注4)。使用新的测试笼和另一只鼠标开始下一个测试。
    7. 录制的视频将被分析,如下所述。


      图1.嗅觉回避测试 A.实验程序的模式;嗅觉回避试验装置。

数据分析

使用录制的视频在10分钟测试期间手动测量冻结和回避时间。 “冻结时间”定义为除呼吸之外,时间保持3秒以上(图2A和视频1)。 “避免时间”被定义为在没有nTMT的香烟的区域内花费的时间,当测试笼分成两个相等的区域时。避免行为由回避指数(避税指数= [P - 50] / 50)表示,其中P是10分钟测试期间回避时间的百分比;图2B)。我们建议通过盲目分析来测量冻结和回避时间。
使用Microsoft Excel通过Welch t 测试来计算价值,其中您单击数据分析并执行 t -test:双样本假设不平等的差异对于多个成对比较,然后根据Holm-Bonferroni方法(Holm,1979)依次评估P 值,以手动方式保持实验α≤0.05。
评估Holm-Bonferroni方法的公式如下:
α/(n-k + 1)
哪里,
n:测试次数,
k:对的排名数。
Holm-Bonferroni校正示例:
考虑具有未调整的P - 值(p 1-4 )的四个零假设(H 1-4),以显着性水平α = 0.05。 H 1:p 1 = 0.01,H 2:p 2 = 0.003,H 3 :p 3 = 0.03,H 4:p 4 = 0.04

  1. 将 P - 值从最小到最小。
    p <2>(= 0.003)&lt; p <1>(= 0.01) p <3>(= 0.03) p 4 (= 0.04)
  2. 为了计算调整后的alpha水平,将Holm-Bonferroni公式用于第一级,并将其与排名第一的P 值(p 2 )进行比较。如果 P 值较小,则拒绝第一排零假设(H 2 )。
    α/(n-k + 1)= 0.05 /(4-1 + 1)= 0.0125
    0.0125 p <2>(= 0.003)。 H 2 被拒绝。
  3. 对第二级重复Holm-Bonferroni公式,并将其与第二级的P 值(p 1 )进行比较。
    α/(n-k + 1)= 0.05 /(4-2 + 1)= 0.0167
    0.0167&gt; p <1>(= 0.01)。 H 1 被拒绝。
  4. 对第三级重复Holm-Bonferroni公式,并将其与排名第三的P 值(p 3 )进行比较。
    α/(n-k + 1)= 0.05 /(4-3 + 1)= 0.0250
    0.0250 < p <3>(= 0.03)。 H <3>不拒绝。
  5. 当您达到第一个未被拒绝的假设时,测试停止。所有后续假设都不重要。我们得出结论,H 1和H 2被拒绝,H 3和H 4不被拒绝。 />

    图2.嗅觉回避试验的样本数据。 A和B.野生型小鼠的冷冻时间(A)和避免指数(B) C.当使用较低量(4μl)的nTMT时,与野生型小鼠相比,5T4敲除小鼠显着降低了冷冻时间(参见Takahashi等人的详细信息, em>。,2016)。

    Video 1. Freezing response in wild-type mice for 40 μl nTMT

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

笔记

  1. 对于这个测试,只能使用鼠标一次,以避免数据混淆,这取决于学习和记忆
  2. 实验者必须限制嗅觉强烈的气味产品,并在测试过程中产生过大的噪音。因为很难彻底清除实验者的个人气味,所以建议在测试之前处理老鼠的同一个实验者在整个测试过程中执行所有的过程。
  3. 因为在这个测试中使用了未稀释的气味,所以防止测试室气味扩散很重要。 nTMT瓶及其衍生物应保存在通风橱中。一次测试完成后,使用nTMT香味的笼子和滤纸应保存在通风橱中。在测试中,首先应使用较少量的nTMT,与较大量相比较。我们建议在专门的室内进行测试,这是不同于习惯的房间,因为防止老鼠在测试前遇到nTMT是很重要的。
  4. 实验者的存在以显着的方式影响小鼠的行为。实验者必须离开测试笼。我们建议当测试阶段从专用测试室开始时,实验者就可以到那里。

致谢

该方案从以前的研究(Kobayakawa等人,2007; Kaneko-Goto等人,2013)中被采用。 (B)(AT),(C)(HT)和创新领域(自适应电路转换)(AT)和挑战性探索性研究(AT)的科学研究资助计划的支持日本教育,文化,体育,科技部(MEXT)。

参考

  1. Holm,S。(1979)。一个简单的顺序拒绝多重测试程序。 Scand J Statist 6(2):65-70。
  2. Kaneko-Goto,T.,Sato,Y.,Katada,S.,Kinameri,E.,Yoshihara,S.,Nishiyori,A.,Kimura,M.,Fujita,H.,Touhara,K.,Reed,RR和Yoshihara,Y。(2013)。 Goofy坐标嗅觉信号的敏锐度。 J Neurosci 33(32):12987-12996。
  3. Kobayakawa,K.,Kobayakawa,R.,Matsumoto,H.,Oka,Y.,Imai,T.,Ikawa,M.,Okabe,M.,Ikeda,T.,Itohara,S.,Kikusui, Mori,K。和Sakano,H。(2007)。小鼠嗅球中的先天性与学习气味处理。 自然 450(7169):503-508。
  4. Takahashi,H.,Ogawa,Y.,Yoshihara,S.,Asahina,R.,Kinoshita,M.,Kitano,T.,Kitsuki,M.,Tatsumi,K.,Okuda,M.,Tatsumi, Wanaka,A.,Hirai,H.,Stern,PL和Tsuboi,A.(2016)。气味检测和歧视行为需要嗅球中间神经元的亚型。 J Neurosci 36(31):8210-8227。 >
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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. Takahashi, H. and Tsuboi, A. (2017). Olfactory Avoidance Test (Mouse). Bio-protocol 7(5): e2153. DOI: 10.21769/BioProtoc.2153.
  2. Takahashi, H., Ogawa, Y., Yoshihara, S., Asahina, R., Kinoshita, M., Kitano, T., Kitsuki, M., Tatsumi, K., Okuda, M., Tatsumi, K., Wanaka, A., Hirai, H., Stern, P. L. and Tsuboi, A. (2016). A subtype of olfactory bulb interneurons is required for odor detection and discrimination behaviors. J Neurosci 36(31): 8210-8227.
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