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Buried Food-seeking Test for the Assessment of Olfactory Detection in Mice
使用寻找掩埋食物试验评估小鼠的嗅觉探测能力   

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The Journal of Neuroscience
Dec 2017

Abstract

The sense of smell allows animals to discriminate a large number of volatile environmental chemicals. Such chemical signaling modulates the behavior of several species that depend on odorant compounds to locate food, recognize territory, predators, and toxic compounds. Olfaction also plays a role in mate choice, mother-infant recognition, and social interaction among members of a group. A key assay to assess the ability to smell odorants is the buried food-seeking test, which checks whether the food-deprived mice can find the food pellet hidden beneath the bedding in the animal’s cage. The main parameter observed in this test is the latency to uncover a small piece of chow, cookie, or other pleasant food, hidden beneath a layer of cage bedding, within a limited amount of time. It is understood that food-restricted mice which fail to use odor cues to locate food within a given time period are likely to have deficits in olfactory abilities. Investigators who used the buried food test, or versions of the buried food test, demonstrated that it is possible to evaluate olfactory deficits in different models of murine studies (Alberts and Galef, 1971; Belluscio et al., 1998; Luo et al., 2002; Li et al., 2013). We have recently used this assay to demonstrate that olfactory-specific Ric-8B knock-out mice (a guanine nucleotide exchange factor that interacts with olfactory-specific G-protein) show an impaired sense of smell (Machado et al., 2017). Here we describe the protocol of the buried food-seeking test, as adopted in our assays.

Keywords: Buried food-seeking test (寻找掩埋食物试验), Ric-8B knock-out mice (Ric-8B敲除小鼠), Olfactory behavior tests (嗅觉行为试验), Olfactory impairment (嗅觉障碍), Olfactory sensory neuron (嗅觉感觉神经元)

Background

The buried food-seeking test was first described in 1971 (Alberts and Galef, 1971). Since then, additional versions of the test have been described. This test has been used to investigate the consequences of olfactory impairment in a variety of situations, such as: analysis of the effects of olfactory function on the performance of female mice in social behavior towards male conspecifics (Yamada et al., 2001), the discrimination of the participation of both the main olfactory system and the vomeronasal organ in behavior (Del Punta et al., 2002) or in animal models of hyposulphataemia, a disturbance in sulphate metabolism (Dawson et al., 2005). It was also used to assess sociability and cognitive function in neuronal cell adhesion molecule (Nrcam) null mice (Moy et al., 2009), to study the effects of the selective non-imidazole histamine H3 receptor antagonist in anxiety and depression-like disorders (Bahi et al., 2014), to analyze the role of endocannabinoids in olfactory sensory neurons (Hutch et al., 2015), and others. There are variations in the buried food test methods used in these studies. For example, some authors used pre-test acclimation in the testing cage to reduce novelty-induced exploratory activity during the olfaction test, while others did not. It is important to note that this acclimation can help in decreasing response variability within groups. In our previous work, we used the buried food test, in association with other motivational, behavioral, and cellular tests, to determinate whether the sense of smell is impaired in olfactory-specific Ric-8B knock-out mice (Machado et al., 2017). The present manuscript describes the protocol of the buried food-seeking test as adopted in our previous assays, in order to observe aspects of olfactory deficits in mice.

Materials and Reagents

  1. Mice should be at least 8-week-old
    Notes:
    1. We used 8-week-old C57BL/6J background mice of both sexes, it is important that the animals have the same age.
    2. It is possible to use this protocol with other strains of mice. It might be applicable to rats as well, but needs standardization of procedures, such as: size of the cage, depth of the bedding, size of the chow pellet, as well as food deprivation time.
    3. In females, estrus can effect olfactory discrimination. Considering this, we initially did statistical analysis in separating genders: wild-type (4 males and 5 females), heterozygote (3 males and 3 females) and conditional knock-out mice (4 males and 5 females). However, we observed no differences between genders, so in our final results, we used both genders in all groups. We recommend evaluating the differences between genders before deciding to use mixed-gender groups or not.
  2. Filtered and autoclaved water
  3. Chow pellets (Food stimulus)
    Notes:
    1. We used a 2 g pellet of the same chow the animals were regularly fed with.
    2. We used AIN-93G chow (for use during rapid growth, pregnancy and lactation, from Rhoster, described in Reeves et al. (1993), because it was the chow type regularly fed to our animals.
    3. Other types of pleasant foods have been previously used as stimulus, such as: Oreo cookie, Kellog’s Fruit Loops, chow covered in peanut butter and other. We found, however, that Kellog’s Fruit Loops did not stimulate foraging through olfactory cues in the mice. In our experiments, regular food chow pellets showed better results in foraging behavior than the Fruit Loops. This may be due to novelty induced hypophagia, so we recommend the use of food stimulus that the animals are accustomed to in order to avoid novelty induced hypophagia.

Equipment

  1. Experimental animal room
    Note: An adequate procedure room is essential for the successful development of the behavior test, since it is sensitive to external distractions. This requirement should be carefully considered during the planning stages. An adequate procedure room consists of an isolated and silent experimentation room, where it is possible to avoid the entrance of people during the test.
  2. Clean mouse cage of a regular size (30.5 cm length x 16 cm width x 16 cm height or similar) (Figure 1) (We used the M.I.C.E.® Animal Care Systems cage [Animal Care Systems, catalog number: M/P 79010 ])
    Note: Do not use regular cage lids. The stainless-steel top which holds food and water interferes with observation and should not be included in the setup. If needed, it is best to use acrylic lids.


    Figure 1. Layout of the buried food-seeking test. The purpose of this experimental test is to measure the animal's ability to use olfactory cues for foraging. The main parameter measured in this test is the latency to find the hidden food. Latency is defined as the time between when the mouse was placed in the cage and when the mouse uncovered the food pellet. For the test, 8-week-old mice were deprived of food for 24 h but received water ad libitum. Next day, a 2 g pellet of regular chow was buried 8 cm beneath the surface of the fresh bedding in one end of a clean test cage. The site of animal placement and the site at which the pellet was buried remained constant.

  3. Fresh cage bedding to create an 8 cm layer in each cage
    Notes:
    1. For each subject, a clean cage washed and dried by the animal care cage washing facility and clean bedding should be used. Do not re-use cages or bedding.
    2. Cage lining: we use Premium Hygienic Animal Bedding LIGNOCEL® FS-14 to bury the pellet.
  4. Portable digital scale (Denver Instrument, model: TR-403 , or equivalent scale)
    Note: To weigh the pellet chow, we used a digital scale (Denver Instrument, TR-403, Max = 410 g, d = 0.001g).
  5. Digital stopwatch
    Note: The stopwatch is used to monitor the test length.
  6. Digital video camera (optional, we used Sony Cyber-shot 14.1 mega pixels) (Sony, model: DSC-W330 )
    Notes:
    1. The presence of a human observer may influence animal behavior. The use of a video camera may help reduce human interference during the test.
    2. All videos were recorded in .avi format and viewed using VLC media player.

Procedure

  1. Deprive 8-week-old mice of food for 24 h: 24 h before the test, remove all chow pellets from the home cage. Check inside the cage to remove any pellet fragments that may be scattered within the cage. Do not remove the water bottle.
    Note: During standardization, we tried using 6-week-old mice and had ambiguous results. 8-week-old mice or older should be used for the behavioral tests because 6-week-old mice are still considered juvenile and show a larger variability in behaviors.
  2. On day 2, it is advisable to acclimate the mouse to the room in the testing cage in the same conditions of the test, before the insertion of the pellet. Acclimation should last as long as the test will last and the animal should be taken out of the cage before the insertion of the pellet.
  3. Initiate the test by burying a 2 g pellet of regular chow 8 cm beneath the surface of the fresh bedding in one of the corners of the test cage. Transfer the mouse from its home cage back into the test cage, in the opposite corner in regard to the pellet. The time from its introduction into the test cage until it finds the food pellet is recorded as the latency (Video 1). The digital stopwatch should be halted as soon as the mouse touches the pellet of chow. If an animal is not able to find the food pellet within 10 min, the test is terminated and the latency is recorded as 600 sec. Importantly, for each subject, a clean cage, clean bedding, and new pellet of chow should be used.

    Video 1. Example of a wild-type mouse foraging during the test. The chow pellet is located in the right corner. The digital stopwatch is halted as soon as the mouse touches the pellet.

  4. It is important to control for motivation in food-seeking behavior. This can be done with either the unburied food-seeking test (Li et al., 2013) or other motivation and locomotion tests (Machado et al., 2017). The unburied food-seeking test is the simplest way to control for motivation. It can be done using either parallel control groups (groups that undergo the unburied food-seeking test at the same time that the test groups undergo the buried food-seeking test) or the same animals used in the buried food-seeking test, after one week. The unburied food seeking test is the same as the buried food-seeking test, with exception to the pellet being on the surface of the bedding, instead of buried. Therefore, this test does not depend on olfactory cues and controls for motivation level among groups.
    Notes:
    1. In the eventuality of an animal escaping the cage during or immediately before the test, exclude this animal from the experiment.
    2. In Machado et al. (2017), while testing for behavioral differences through olfactory impairment, three groups were used: the test-group (conditional knock-out, n = 9) and two control groups (wild-type, n = 9; and heterozygote mice, n = 6). The test-group showed longer latency in retrieving the food pellet, in comparison to the control-groups (WT 143.1 ± 30.2 sec; Het 188.3 ± 24.5 sec; cKO 378.5 ± 76.3 sec; p = 0.02, F(2,15) = 4.54, one-way ANOVA and Dunnett post-test). It is important to note that in this article, we acclimatized the mice to the experimentation room, but not to the cage. It is possible that further acclimation to the cage may render more intensely different latencies between groups (Figure 2). Additionally, in this study we did not perform the unburied food-seeking test because we evaluated motivation and locomotion through the open-field, light/dark, elevated plus-maze, and tail suspension tests.
    3. In order to evaluate possible differences between male and female mice, the three groups used in Machado et al. (2017) were initially divided in genders: wild-type (4 males and 5 females), heterozygote (3 males and 3 females) and conditional knock-out mice (4 males and 5 females). However, after initial statistical analysis, we observed no differences between genders, so the final results contain both genders in each group.


      Figure 2. Buried food-seeking test results. Ric-8b conditional knock-out (cKO) mice required significantly longer times to find the food. WT (n = 9), Het (n = 6), and cKO (n = 9) (extracted from Machado et al., 2017).

Data analysis

  1. For experimental design, at least three animals of each group test should be used.
  2. The time necessary for the animal to retrieve the pellet (latency) was measured in seconds up to a maximum of 10 min (600 sec was the maximum score).
  3. Any preferred statistical program can be used for statistical analysis. To compare two data sets, use Student’s t-test. To compare three data sets or more, use One-way ANOVA and post-hoc Dunnett or similar. For not normal distribution of data or small groups, use Mann-Whitney test to replace the Student’s t-test and Kruskal-Wallis in replace for one-way ANOVA. Differences are considered significant at P ≤ 0.05.

Notes

  1. The score on a behavioral test can be influenced by many factors, such as lab temperature, noise and the animal’s circadian rhythm. In this way, the experiments should always be done at the same time of day, always respecting the animal's circadian cycle, in an isolated and silent experimentation room, avoiding the entrance of people during the test. It is highly recommended to acclimate the animals in a separate room dedicated specifically for acclimation; this room should be similar to the testing room.
  2. A single experimenter should complete an experiment in its entirety, to reduce variability. Importantly, if multiple cohorts of mice are used, it is preferable that the same experimenter conduct testing on all the mice in all the cohorts of the study to reduce variability.
  3. We recommend double-blind tests. The experimenter that does the video analysis should be blind to the experimental groups in order to avoid bias.

Acknowledgments

This work was supported by Grants from Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP; 2016/24471-0 (B.M.), and 2012/24640-6 (C.F.M.)]. We thank Silvânia S. P. Neves, Renata Spalutto Fontes, and Flávia de Moura Prates Ong for technical assistance in our animal facility. This protocol was adapted from previous works (Alberts and Galef, 1971; Li et al., 2013). The authors declare no competing financial interests.

References

  1. Alberts, J. R. and Galef, B. G., Jr. (1971). Acute anosmia in the rat: a behavioral test of a peripherally-induced olfactory deficit. Physiol Behav 6(5): 619-621.
  2. Bahi, A., Schwed, J. S., Walter, M., Stark, H. and Sadek, B. (2014). Anxiolytic and antidepressant-like activities of the novel and potent non-imidazole histamine H3 receptor antagonist ST-1283. Drug Des Devel Ther 8: 627-637.
  3. Belluscio, L., Gold, G. H., Nemes, A. and Axel, R. (1998). Mice deficient in Golf are anosmic. Neuron 20(1): 69-81.
  4. Dawson, P. A., Steane, S. E. and Markovich, D. (2005). Impaired memory and olfactory performance in NaSi-1 sulphate transporter deficient mice. Behav Brain Res 159(1): 15-20.
  5. Del Punta, K., Leinders-Zufall, T., Rodriguez, I., Jukam, D., Wysocki, C. J., Ogawa, S., Zufall, F. and Mombaerts, P. (2002). Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes. Nature 419(6902): 70-74.
  6. Hutch, C. R., Hillard, C. J., Jia, C. and Hegg, C. C. (2015). An endocannabinoid system is present in the mouse olfactory epithelium but does not modulate olfaction. Neuroscience 300: 539-553.
  7. Li, F., Ponissery-Saidu, S., Yee, K. K., Wang, H., Chen, M. L., Iguchi, N., Zhang, G., Jiang, P., Reisert, J. and Huang, L. (2013). Heterotrimeric G protein subunit Gγ13 is critical to olfaction. J Neurosci 33(18): 7975-7984.
  8. Luo, A. H., Cannon, E. H., Wekesa, K. S., Lyman, R. F., Vandenbergh, J. G. and Anholt, R. R. (2002). Impaired olfactory behavior in mice deficient in the α subunit of Go. Brain Res 941(1-2): 62-71.
  9. Machado, C. F., Nagai, M. H., Lyra, C. S., Reis-Silva, T. M., Xavier, A. M., Glezer, I., Felicio, L. F. and Malnic, B. (2017). Conditional deletion of Ric-8b in olfactory sensory neurons leads to olfactory impairment. J Neurosci 37(50): 12202-12213.
  10. Moy, S. S., Nonneman, R. J., Young, N. B., Demyanenko, G. P. and Maness, P. F. (2009). Impaired sociability and cognitive function in Nrcam-null mice. Behav Brain Res 205(1): 123-131.
  11. Reeves, P. G., Nielsen, F. H. and Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123(11): 1939-1951.
  12. Yamada, K., Wada, E. and Wada, K. (2001). Female gastrin-releasing peptide receptor (GRP-R)-deficient mice exhibit altered social preference for male conspecifics: implications for GRP/GRP-R modulation of GABAergic function. Brain Res 894(2): 281-287.
Figure 2. Buried food-seeking test result

简介

嗅觉允许动物区分大量挥发性环境化学物质。这种化学信号调节了几种依靠气味化合物定位食物,识别领土,捕食者和有毒化合物的物种的行为。嗅觉也在配偶选择,母婴认知以及群体成员之间的社交互动中发挥作用。评估嗅觉气味能力的关键检测方法是埋藏食物追踪测试,该测试检查食物剥夺的老鼠是否可以在动物的笼子中找到隐藏在寝具下面的食物小球。在这个测试中观察到的主要参数是在有限的时间内发现一小块食物,饼干或其他令人愉快的食物,隐藏在一层笼子底座之下的延迟。据了解,在特定时间内不能使用气味提示来定位食物的食物受限小鼠可能具有嗅觉能力缺陷。使用掩埋食物测试或埋藏食物测试版本的调查人员证明,可以在不同模型的小鼠研究中评估嗅觉缺陷(Alberts和Galef,1971; Belluscio等人,, 1998; Luo等人,2002; Li等人,2013)。我们最近使用该测定来证明嗅觉特异性Ric-8B敲除小鼠(与嗅觉特异性G蛋白相互作用的鸟嘌呤核苷酸交换因子)显示出嗅觉受损(Machado等人, ,2017)。在这里,我们描述了我们分析中采用的掩埋食物寻求测试的协议。

【背景】1971年首次描述了埋藏食物寻求试验(Alberts和Galef,1971)。从那以后,已经描述了其他版本的测试。该测试已被用于研究各种情况下嗅觉障碍的后果,例如:分析嗅觉功能对雌性小鼠在针对男性同种群的社交行为中的表现的影响(Yamada等人, ,2001),主要嗅觉系统和犁鼻器官在行为上的参与的区分(Del Punta et al。,2002),或硫酸亚胺血症的动物模型,硫酸盐紊乱代谢(Dawson等人,2005)。它也被用于评估神经元细胞粘附分子(Nrcam)缺失小鼠(Moy等,2009)中的社交性和认知功能,以研究选择性非咪唑组胺H (Bahi等人,2014),以分析内源性大麻素在嗅觉感觉神经元中的作用(Hutch等人, ,2015)等。这些研究中使用的掩埋食物测试方法有所不同。例如,一些作者在测试笼中使用预测试适应来减少嗅觉测试期间由新奇引发的探索活动,而另一些则没有。重要的是要注意,这种适应能够帮助减少组内的响应变化。在我们以前的工作中,我们使用掩埋食物测试,结合其他动机,行为和细胞测试来确定嗅觉特异性Ric-8B敲除小鼠中的嗅觉是否受损(Machado等人,2017)。本手稿描述了我们以前测定中采用的掩埋食物寻求测试的方案,以观察小鼠中嗅觉缺陷的方面。

关键字:寻找掩埋食物试验, Ric-8B敲除小鼠, 嗅觉行为试验, 嗅觉障碍, 嗅觉感觉神经元

材料和试剂

  1. 小鼠应至少8周龄
    注意:
    1. 我们使用了8周龄的两性C57BL / 6J背景小鼠,重要的是这些动物的年龄相同。
    2. 有可能将这种方案与其他的小鼠品系一起使用。它也可能适用于老鼠,但需要标准化程序,例如:笼子的大小,床铺的深度,食物颗粒的大小以及食物剥夺时间。
    3. 在雌性动物中,发情可以影响嗅觉辨别。考虑到这一点,我们最初对分离性别进行了统计分析:野生型(男性4例,女性5例),杂合子(男性3例,女性3例)和条件性敲除小鼠(男性4例,女性5例)。然而,我们没有观察到性别之间的差异,所以在我们的最终结果中,我们在所有组中都使用了两个性别。我们建议您在决定是否使用混合性别群组之前评估性别之间的差异。
  2. 过滤和蒸压水
  3. 食物颗粒(食物刺激)
    注意:
    1. 我们使用了与动物经常喂食的相同食物的2g颗粒。
    2. 我们使用AIN-93G食物(用于Reeves等人(1993)描述的来自Rhoster的快速生长,怀孕和哺乳期,因为它是经常喂给我们动物的食物类型。 br />
    3. 其他类型的宜人食物以前曾被用作刺激物,例如:奥利奥饼干,凯洛格的水果环,食用花生酱和其他食物。然而,我们发现凯洛格的水果圈没有刺激老鼠通过嗅觉线索觅食。在我们的实验中,普通食物饲料丸在觅食行为上比水果环显示出更好的结果。这可能是由于新奇引起的食欲不振,所以我们建议使用动物习惯的食物刺激,以避免新奇引起的食欲不振。

设备

  1. 实验动物房
    注:对于行为测试的成功开发来说,充足的手术室是必不可少的,因为它对外部干扰很敏感。在规划阶段应该认真考虑这一要求。一个足够的手术室由一个孤立的无声实验室组成,在这个实验室里可以避免测试过程中人员进入。
  2. 清洁常规尺寸(30.5厘米长×16厘米宽×16厘米高或类似尺寸)的鼠笼(图1)[我们使用MICE 动物护理系统笼(动物护理系统,目录号:M / P 79010)。]
    注意:不要使用普通的笼盖。容纳食物和水的不锈钢顶部会干扰观察,不应包含在设置中。如果需要,最好使用丙烯酸盖。


    图1.掩埋食物寻求测试的布局这个实验测试的目的是测量动物嗅觉线索觅食的能力。在这个测试中测量的主要参数是查找隐藏食物的延迟。延迟被定义为鼠标放入笼子中和鼠标揭开食物颗粒之间的时间。为了测试,8周龄的小鼠被剥夺了食物24小时,但接受了水随意 。第二天,在清洁试验笼的一端,将新鲜寝具表面下8厘米埋入2克普通食物丸。
    。动物的安置地点和沉淀的地点保持不变。

  3. 新鲜的笼子被褥在每个笼子里创造一个8厘米的层。
    备注:
    1. 对于每个受试者,都应该使用一个清洁的笼子,由动物护理笼子清洗设施进行清洗和干燥,并使用干净的床上用品。请勿重复使用笼子或寝具。
    2. 笼子内衬:我们使用优质卫生动物床垫LIGNOCEL ® FS-14来掩埋药丸。
  4. 便携式数字秤(丹佛仪器,型号:TR-403,或同等规模)
    注意:为了称量颗粒食物,我们使用了数字刻度(丹佛仪器,TR-403,最大= 410克,d = 0.001克)。
  5. 数码秒表
    注意:秒表用于监视测试长度。
  6. 数码摄像机(可选,我们使用Sony Cyber-shot 14.1百万像素)(Sony,型号:DSC-W330)
    注意:
    1. 人类观察者的存在可能会影响动物的行为。使用摄像机可能有助于减少测试过程中的人为干扰。
    2. 所有视频均以.avi格式录制,并使用VLC媒体播放器查看

程序

  1. 在试验前24小时剥夺8周龄食物老鼠24小时,从家笼中取出所有食物颗粒。检查笼内部以去除可能在笼内散落的任何颗粒碎片。不要取下水瓶。
    注意:在标准化期间,我们尝试使用6周龄的小鼠,结果不明确。 8周龄或更老的小鼠应该用于行为测试,因为6周龄的小鼠仍然被认为是少年,并且行为变化更大。
  2. 在第2天,在插入颗粒之前,在相同的测试条件下,将鼠标适应试验笼中的房间是明智的。驯化应持续,只要测试将持续,在插入颗粒之前应将动物从笼中取出。
  3. 通过在测试笼的一个角落中在新鲜床上用品的表面下方埋入8克常规食物的2g颗粒开始测试。将鼠标从其家笼转移回测试笼中,在对角处的颗粒上。从引入到测试笼直到发现食物颗粒的时间被记录为潜伏期(视频1)。只要鼠标碰到食物的颗粒,数字秒表应该停止。如果动物在10分钟内无法找到食物颗粒,则停止试验并记录等待时间为600秒。重要的是,对于每个主题,都应该使用干净的笼子,干净的床上用品和新的丸子。

    视频1
  4. 控制寻找食物行为的动机非常重要。这可以通过未掩埋食物寻求测试(Li et al。,2013)或其他动机和运动测试(Machado et al。,2017)来完成。未掩埋的食物追求测试是控制动机的最简单方法。可以使用平行对照组(在测试组进行埋藏食物寻求测试的同时进行未掩埋食物寻求测试的组)或埋藏食物寻求测试中使用的相同动物周。未埋藏的食物寻找测试与掩埋的食物寻找测试相同,除了沉淀物在表面上而不是掩埋之外。因此,这个测试不依赖于嗅觉线索和组间动机水平的控制。
    注意:
    1. 在试验期间或即将进行试验的动物逃避笼子的可能性中,将该动物排除在实验之外。
    2. 在Machado et al。 (2017),同时通过嗅觉障碍测试行为差异,使用三组:测试组(有条件的敲除,n = 9)和两个对照组(野生型,n = 9;和杂合子小鼠,n = 6)。与对照组相比,测试组显示了回收食物颗粒的更长的潜伏期(WT 143.1±30.2秒; Het 188.3±24.5秒; cKO 378.5±76.3秒; p = 0.02,F < (2,15) = 4.54,单因素方差分析和Dunnett事后检验)。值得注意的是,在这篇文章中,我们使老鼠适应实验室,但不适合笼子。进一步适应笼子可能会导致组间潜伏期更加剧烈(图2)。此外,在这项研究中,我们没有进行未掩埋的食物寻求测试,因为我们通过开放式场地,明/暗,高架十字迷宫和尾悬挂测试来评估动机和运动。
    3. 为了评估雄性和雌性小鼠之间的可能差异,在Machado等人中使用的三个组(2017年)最初分为两类:野生型(男性4例,女性5例),杂合子(3例男性,女性3例)和条件性敲除小鼠(男性4例,女性5例)。然而,经过最初的统计分析后,我们观察到性别之间没有差异,因此最终结果包含每个组别的两个性别。


    图2.埋藏的食物寻求测试结果Ric-8b有条件敲除(cKO)小鼠需要明显更长的时间才能找到食物。 WT(n = 9),Het(n = 6)和cKO(n = 9)(提取自Machado等人,2017)。

数据分析

  1. 对于实验设计,应该使用每组试验中至少三只动物。

  2. 动物取回药丸所需的时间(潜伏期)以秒为单位进行测量,最长可达10分钟(最长得分为600秒)。
  3. 任何优选的统计程序都可以用于统计分析。要比较两个数据集,请使用Student's t -test。要比较三个或更多的数据集,请使用单向方差分析和事后Dunnett或类似方法。对于数据或小组的不正态分布,使用Mann-Whitney检验来代替Student's检验和Kruskal-Wallis替代单因素方差分析。 P ≤0.05时,差异被认为是显着的。

笔记

  1. 行为测试的得分可能受许多因素影响,如实验室温度,噪音和动物的昼夜节律。通过这种方式,实验应始终在一天中的同一时间进行,并始终尊重动物的昼夜节律周期,在一个孤立且无声的实验室内进行,避免测试过程中人员进入。强烈建议在适合专门适应环境的独立房间内适应动物;这个房间应该与测试室相似。
  2. 一个实验者应该完整地完成一个实验,以减少变异。重要的是,如果使用多组小鼠,优选的是相同的实验者对所有研究组中的所有小鼠进行测试以减少变异性。
  3. 我们建议进行双盲测试。
    视频分析的实验者应该对实验组视而不见,以避免偏见。

致谢

这项工作得到了Fundaçãode AmparoàPesquisa do Estado deSãoPaulo [FAPESP; 2016 / 24471-0(B.M.)和2012 / 24640-6(C.F.M.)]。我们感谢SilvâniaS. P. Neves,Renata Spalutto Fontes和Fláviade Moura Prates Ong在我们的动物设施中提供技术援助。该协议改编自以前的作品(Alberts和Galef,1971; Li等人,2013年)。作者声明没有竞争的财务利益。

参考

  1. Alberts,J.R。和Galef,B.G.,Jr.(1971)。 大鼠急性嗅觉缺失:外周诱发的嗅觉缺陷的行为测试。 Physiol Behav 6(5):619-621。
  2. Bahi,A.,Schwed,J. S.,Walter,M.,Stark,H.和Sadek,B。(2014)。 新型有效非咪唑组胺H 3的抗焦虑和抗抑郁药样活性,受体拮抗剂ST-1283。 Devel Ther 8:627-637。
  3. Belluscio,L.,Gold,G. H.,Nemes,A。和Axel,R。(1998)。 G olf 缺陷的小鼠是无神论的。 Neuron 20(1):69-81。
  4. Dawson,P.A.,Steane,S.E。和Markovich,D。(2005)。 NaS 1 -1硫酸盐转运体缺陷小鼠的记忆和嗅觉功能受损。 Behav Brain Res 159(1):15-20。
  5. Del Punta,K.,Leinders-Zufall,T.,Rodriguez,I.,Jukam,D.,Wysocki,C.J.,Ogawa,S.,Zufall,F.and Mombaerts,P.(2002)。 缺乏一簇犁鼻器受体基因的小鼠中缺乏信息素应答 Nature 419(6902):70-74。
  6. Hutch,C. R.,Hillard,C. J.,Jia,C.和Hegg,C. C.(2015)。 内源性大麻素系统存在于小鼠嗅觉上皮中,但不会调节嗅觉。 Neuroscience 300:539-553。
  7. Li,F.,Ponissery-Saidu,S.,Yee,KK,Wang,H.,Chen,ML,Iguchi,N.,Zhang,G.,Jiang,P.,Reisert,J.and Huang, 2013)。 异三聚体G蛋白亚基Gγ13对嗅觉是至关重要的。 J Neurosci 33(18):7975-7984。
  8. Luo,A.H.,Cannon,E.H。,Wekesa,K.S.,Lyman,R.F.,Vandenbergh,J.G。和Anholt,R.R。(2002)。 在缺乏G亚α亚基的小鼠中的嗅觉行为受损。 Brain Res 941(1-2):62-71。
  9. Machado,C. F.,Nagai,M. H.,Lyra,C. S.,Reis-Silva,T. M.,Xavier,A. M.,Glezer,I.,Felicio,L. F.和Malnic,B。(2017)。 在嗅觉感觉神经元中条件性删除Ric-8b会导致嗅觉障碍。 J Neurosci 37(50):12202-12213。
  10. Moy,S.S.,Nonneman,R.J.,Young,N.B。,Demyanenko,G.P.和Maness,P.F。(2009)。 Nrcam-null小鼠的社交能力和认知功能受损行为研究Brain Res 205(1):123-131。
  11. Reeves,P.G.,Nielsen,F.H。和Fahey,G.C.,Jr.(1993)。 AIN-93实验室啮齿动物纯化饮食:美国营养学会特设写作委员会最终报告关于AIN-76A啮齿类动物饮食的改写。 Nutr 123(11):1939-1951。
  12. Yamada,K.,Wada,E.和Wada,K。(2001)。 雌性胃泌素释放肽受体(GRP-R)缺陷型小鼠表现出对男性同胞的社会偏好改变:GRP / GRP-R调节GABA能功能的意义。 Brain Res 894(2):281-287。
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Copyright: © 2018 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. Machado, C. F., Reis-Silva, T. M., Lyra, C. S., Felicio, L. F. and Malnic, B. (2018). Buried Food-seeking Test for the Assessment of Olfactory Detection in Mice. Bio-protocol 8(12): e2897. DOI: 10.21769/BioProtoc.2897.
  2. Machado, C. F., Nagai, M. H., Lyra, C. S., Reis-Silva, T. M., Xavier, A. M., Glezer, I., Felicio, L. F. and Malnic, B. (2017). Conditional deletion of Ric-8b in olfactory sensory neurons leads to olfactory impairment. J Neurosci 37(50): 12202-12213.
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