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Olfactory Habituation-dishabituation Test (Mouse)
嗅觉习惯化-去习惯化试验(小鼠)   

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Abstract

Olfaction plays a fundamental role in the various behaviors such as feeding, mating, nursing, and avoidance in mice. Behavioral tests that characterize 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 habituation-dishabituation test for investigating the odor detection threshold in mice.

Keywords: Olfaction(嗅觉), Odor detection threshold(气味检测阈值), Behavioral test(行为测试), Mouse(小鼠), Olfactometer(嗅觉计)

Background

Olfactory system is a good model for studying the sensory processing in the brain. To characterize abilities of odor detection in genetically modified mice, the olfactory habituation-dishabituation test was performed using either filter paper or cotton scented with a test odor (Kobayakawa et al., 2007; Ferquson et al., 2000). There is a natural tendency of mice preferentially exploring novelty such as a novel odor and a novel object (Bevins and Besheer, 2006). Therefore, there is no training in advance before starting these tests. However, it is difficult to constantly supply the test odor especially at lower concentrations (around the detection threshold), because the odor is diluted by diffusion during the test. Here, we describe a method for the olfactory habituation-dishabituation test using an olfactometer. This method has an advantage in the point that mice are exposed to the odor at a constant concentration during the test, compared with that using the filter paper (Takahashi et al., 2016).

Materials and Reagents

  1. Latex gloves (NIPPON Genetics, catalog number: SLPF-M )
  2. Paper towels (KCWW, Kimberly-Clark, catalog number: 47000 )
  3. Filter tips
    10 μl (FUKAEKASEI and WATSON, catalog number: 1252-207CS )
    200 μl (FUKAEKASEI and WATSON, catalog number: 1252-703CS )
    1,000 μl (FUKAEKASEI and WATSON, catalog number: 124-1000S )
  4. Laboratory-bred mice
    Note: 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. Eugenol (6.3 M) (Nacalai Tesque, catalog number: 15806-42 )
  6. 70% ethanol

Equipment

  1. Olfactometer with two channels (Matsumi Kagaku Keisoku)
  2. Gas cylinder (normal air)
  3. Test cage (25 x 37 x 24 cm, polypropylene)
  4. Clear acrylic board (which can cover the roof of the test cage)
  5. 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.
  6. Tripod for camera

Note: The gas cylinder containing clean air is connected to the olfactometer. The olfactometer regulates the gas flow (0.5 L/min), and switches flow between clean air and air with an odor by passing the air through a bottle containing eugenol. The olfactometer, connected to the test cage (25 x 37 x 24 cm), can supply either the clean air or the odor through the gas port (0.5-mm diameter hole) on the wall (2-cm height). The roof of test cage is covered with clear acrylic board (Figure 1).


Figure 1. Apparatus of the olfactory habituation-dishabituation test

Software

  1. Microsoft Excel (Microsoft)

Procedure

  1. Before the test day
    1. For this test, mice should be used only once to avoid confounding of data, based on the learning and memory. During five days before the test, mice are habituated to the experimental condition. The same experimenter gently handles each mouse with latex gloves on for 10 min per day during the five consecutive days in the experimental room.

  2. On the test day
    1. Tests are done during the light phase of 12 h light/dark cycle in the test room, in which, the weak light (< 5 lux) is used for indirect lighting. Prepare a series of odor solutions diluted with deionized water (e.g., 10-4, 10-5, 10-6 and 10-7 in the case of eugenol), and set up the apparatus described in Equipment section in the test room (Figure 1).
    2. The mouse is transferred from their home cage to the test cage.
    3. Supply the clean air to the test cage, to which have been habituated for 15 min (Figure 2A; habituation). Because the mice often excrete their feces and urine several times in the test cage, especially in the beginning of habituation phase, clean the floor using paper towels every 5 min (three times) to prevent such odors from diffusion in the test cage.
    4. Continuously supply the clean air to the test cage for 3 min (Figure 2A; 1st trial). Record the mouse behavior with a digital video camera during the 1st and 2nd trials.
    5. Switch the air flow and the mice are exposed to the odor for 3 min (Figure 2A; 2nd trial).
    6. The tested mouse is returned to their home cage. Carefully clean the test cage using paper towels with 70% ethanol. Supply the clean air for 5 min to the test cage to clear the odor from the connecting tube between the olfactometer and test cage. Start the next test using another mouse.
    7. The recorded videos are analyzed, as described below.


      Figure 2. Schema of the experimental procedure. A. Schema of the experimental procedure; B. Investigation time is defined as the time when the nose entered the 2.5-cm square area near the gas port (blue dotted line).

Data analysis

Investigation times in the 1st and 2nd trials are measured manually during the 3-min test using the recorded videos. ‘Investigation time’ is defined as the time when the nose enters the 2.5-cm square area near the gas port (Figure 2B and Video 1). We recommend that the investigation times are measured by blinded analysis. Differences in investigation times between the 1st trial and 2nd trial are calculated as the mean ± SEM (Figures 3A and 3B).
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 = 0.005, H3: p3 = 0.03, H4: p4 = 0.04

  1. Order the P-values from smallest to largest.
    p2 (= 0.005) < 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 (= 0.005). 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 3. Sample data of the habituation-dishabituation test. A. Investigation time in the 1st and 2nd trials using 6.3 μM eugenol in wild-type mice. B. Differences in investigation times between the 1st and 2nd trials using different concentrations of eugenol in wild-type mice and 5T4 knockout (KO) mice, in which the 5T4 coding region was replaced by LacZ (Southgate et al., 2010). A single-pass transmembrane glycoprotein, 5T4, is known to regulate the dendritic development of olfactory bulb interneurons. At 6.3 μM, the investigation time of 5T4 KO mice was remarkably shorter than that of wild-type mice. These results suggest that a subset of olfactory bulb interneurons is required for the odor detection behavior (see the detail in Takahashi et al., 2016).

    Video 1. Response of wild-type mice to 6.3 μM eugenol

Notes

  1. For the test, male mice should be used to avoid the effect of the estrous cycle.
  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. Prepare a series of odor solutions with dilutions outside the test room using filter tips (Procedure, step B1). In the test, a low concentration of eugenol should be used first, compared with the high concentration.
  4. The existence of experimenter affects the mouse behaviors in no small 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 modified from a previous study (Kobayakawa et al., 2007). 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. Bevins, R. A. and Besheer, J. (2006). Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ‘recognition memory’. Nat Protoc 1(3): 1306-1311.
  2. Ferguson, J. N., Young, L. J., Hearn, E. F., Matzuk, M. M., Insel, T. R. and Winslow, J. T. (2000). Social amnesia in mice lacking the oxytocin gene. Nat Genet 25(3): 284-288.
  3. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scand J Statist 6(2): 65–70.
  4. 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.
  5. Southgate, T. D., McGinn, O. J., Castro, F. V., Rutkowski, A. J., Al-Muftah, M., Marinov, G., Smethurst, G. J., Shaw, D., Ward, C. M., Miller, C. J. and Stern, P. L. (2010). CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells. PLoS One 5(4): e9982.
  6. 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

嗅觉对小鼠的喂养,交配,护理和避孕等各种行为起着至关重要的作用。 使用遗传修饰的小鼠表征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,我们描述了嗅觉习惯 - 适应性测试,用于调查小鼠的气味检测阈值。
【背景】嗅觉系统是研究大脑感觉处理的好模型。为了表征遗传修饰小鼠中气味检测的能力,使用具有测试气味的滤纸或棉花香气进行嗅觉习惯 - 抹杀测试(Kobayakawa et al。,2007; Ferquson等,2000)。有一种自然的倾向,老鼠优先探索新奇,如新颖的气味和一个新的对象(Bevins和Besheer,2006)。因此,在开始这些测试之前,没有提前进行培训。然而,由于在试验期间通过扩散稀释气味,所以难以不断地提供测试气味,特别是在较低的浓度(检测阈值附近)。在这里,我们描述一种使用嗅觉仪进行嗅觉习惯 - 消食抑制测试的方法。与使用滤纸(Takahashi等人,2016)相比,该方法的优点在于,在测试期间,小鼠以恒定的浓度暴露于气味。

关键字:嗅觉, 气味检测阈值, 行为测试, 小鼠, 嗅觉计

材料和试剂

  1. 乳胶手套(NIPPON Genetics,目录号:SLPF-M)
  2. 纸巾(KCWW,Kimberly-Clark,目录号:47000)
  3. 过滤提示
    10μl(FUKAEKASEI和WATSON,目录号:1252-207CS)
    200μl(FUKAEKASEI和WATSON,目录号:1252-703CS)
    1,000μl(FUKAEKASEI和WATSON,目录号:124-1000S)
  4. 实验室繁殖小鼠
    注意:小鼠按每个笼子3-5个容纳,保存在具有受控温度(〜23℃)和12小时光/黑暗循环(8:30 AM点亮)的湿度的空间中,随意获得食物和水。
  5. 丁香酚(6.3M)(Nacalai Tesque,目录号:15806-42)
  6. 70%乙醇

设备

  1. 嗅觉仪表有两个通道(松井久久Ke)
  2. 气瓶(正常空气)
  3. 测试笼(25 x 37 x 24厘米,聚丙烯)
  4. 透明丙烯酸板(可覆盖测试笼的屋顶)
  5. 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。
  6. 三脚架相机

注意:含有清洁空气的气瓶连接到嗅觉计。嗅觉仪调节气流(0.5升/分钟),并通过将空气通过含有丁香酚的瓶子,在洁净空气和空气之间切换气流。连接到测试笼(25 x 37 x 24厘米)的嗅觉仪可以通过墙壁上的气体口(0.5毫米直径的孔)(2厘米高度)提供清洁的空气或气味。测试笼的屋顶覆盖有透明的亚克力板(图1)。


图1.嗅觉习惯 - 抑郁测试的装置

软件

  1. Microsoft Excel(Microsoft)

程序

  1. 考试前一天
    1. 对于这个测试,应该仅使用一次鼠,以避免数据混淆,这取决于学习和记忆。在测试前五天,小鼠习惯于实验条件。实验室在实验室连续五天内,每天用乳胶手套轻轻处理每只小鼠10分钟。

  2. 在考试当天
    1. 在测试室中的12小时光/暗循环的光照期间进行测试,其中弱光(<5勒克斯)用于间接照明。制备一系列用去离子水稀释的气味溶液(例如,10℃-4℃,10℃-5℃,10℃-6℃,/sup>和10 -7 ),并设置在试验室的设备部分中描述的装置(图1)。
    2. 鼠标从他们的家笼转移到测试笼。
    3. 为测试笼提供清洁空气,已经习惯了15分钟(图2A;习惯)。因为小鼠经常在测试笼中排出粪便和尿液多次,特别是在习惯阶段开始时,每隔5分钟(三次)用纸巾清洁地板,以防止这些气味在测试笼中扩散。 />
    4. 将清洁空气连续供给测试笼3分钟(图2A;第一次试验)。在第一次和第二次试验中,使用数码摄像机记录鼠标行为。
    5. 切换气流,小鼠暴露于气味3分钟(图2A;第二次试验)。
    6. 被测试的鼠标返回到他们的家笼。用70%乙醇的纸巾小心清洁测试笼。向测试笼提供清洁空气5分钟,以清除嗅觉计和测试笼之间连接管的气味。使用另一只鼠标开始下一次测试。
    7. 录制的视频将被分析,如下所述。


      图2.实验程序的模式。 A.实验程序的模式; B.调查时间定义为鼻子进入靠近气体口的2.5厘米正方形区域(蓝色虚线)的时间。

数据分析

使用录制的视频,在3分钟测试期间手动测量第一和第二次试验中的调查时间。 "调查时间"定义为鼻子进入气体口附近的2.5厘米正方形区域的时间(图2B和视频1)。我们建议调查时间通过盲目分析来衡量。第一次试验和第二次试验之间的调查时间差异以平均值±SEM计算(图3A和3B)。
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:H 2:p 2 = 0.005,H 3:p 1 3 = 0.03,H 4:p <4> = 0.04

  1. P - 值从最小到最小。
    p <2>(= 0.005) 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>(= 0.005)。 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> 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3.习惯性 - 耐受性测试的样本数据。 A.在野生型小鼠中使用6.3μM丁香酚的第一次和第二次试验中的调查时间。 B.在野生型小鼠和5T4敲除(KO)小鼠中使用不同浓度的丁子香酚的第一次和第二次试验之间的调查时间差异,其中5T4编码区被LacZ (Southgate等人,2010)所取代。已知一种单程跨膜糖蛋白5T4调节嗅球中间神经元的树突状发育。在6.3μM,5T4 KO小鼠的调查时间明显短于野生型小鼠。这些结果表明,气味检测行为需要嗅球中间神经元的一个子集(参见高桥等人的详细信息,2016)。

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笔记

  1. 对于测试,应使用雄性小鼠避免发情周期的影响
  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。使用过滤嘴在试验室外准备一系列稀释的气味溶液(步骤B1)。在测试中,首先应该使用低浓度的丁子香酚,与高浓度相比。
  4. 实验者的存在不会影响小鼠的行为。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

该协议从先前的研究(Kobayakawa等人,2007)修改。 (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)。

参考文献

  1. Bevins,RA和Besheer,J。(2006)。  大鼠和小鼠中的对象识别:用于研究"识别记忆"的单试验非匹配样本学习任务。 Nat Protoc 1(3):1306-1311。 br />
  2. Ferguson,JN,Young,LJ,Hearn,EF,Matzuk,MM,Insel,TR和Winslow,JT(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/10888874"target ="_ blank">缺乏催产素基因的小鼠中的社会遗忘。Nat Genet 25(3):284-288。
  3. 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。
  4. 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。
  5. Southgate,TD,McGinn,OJ,Castro,FV,Rutkowski,AJ,Al-Muftah,M.,Marinov,G.,Smethurst,GJ,Shaw,D.,Ward,CM,Miller,CJ and Stern,PL(2010 )。 CXCR4介导的趋化性受5T4蛋白糖蛋白调控小鼠胚胎细胞。 PLoS One 5(4):e9982。
  6. 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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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 Habituation-dishabituation Test (Mouse). Bio-protocol 7(5): e2154. DOI: 10.21769/BioProtoc.2154.
  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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