Olfactory Habituation in Fasted Mice

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Nature Neuroscience
Mar 2014



Sensory perception is tightly modulated by the individual’s internal states. In particular, it has been shown that olfactory processes are constantly influenced by metabolic signals reflecting the energy status of the body. Thus, it is important to implement novel approaches to evaluate the impact of body energy changes on olfactory performance. Here, we describe a behavioral protocol to accurately evaluate olfactory habituation in fasted mice (Soria-Gomez et al., 2014) using basic equipment that mice are familiar with. Briefly, the mouse is placed in a test cage where it is presented first, an odorless solvent (the control), then an odor A (twice) and finally an odor B. This test relies on the fact that animals present an attenuation of the behavioral response after several presentations of the same olfactory stimulus.

Keywords: Food intake (食物的摄入量), Fasting (禁食的), Mice (老鼠), Olfactory habituation (嗅觉的习惯), Olfactory test (嗅觉测试)

Materials and Reagents

  1. Animals: C57/BL6N male mice (Janvier Labs)
  2. Mineral oil (Sigma-Aldrich, catalog number: M-5904 )
  3. Isoamyl acetate (banana odor) (Sigma-Aldrich, catalog number: W205508 )
  4. Benzaldehyde (almond odor) (Sigma-Aldrich, catalog number: 418099 )
  5. 20% ethanol
    Note: Mineral oil was used as a solvent, because of its odorless property as reported in the literature (Linster et al., 2009; Qiu et al., 2014; Slotnick and Restrepo, 2005; Tan et al., 2010). Almond and banana odors were used as novel, yet neutral odors(Yang et al., 2013; Yang and Crawley, 2009). However, other odors are also suitable to test in our conditions, such as hexanal.


  1. Standard individual plexiglass cage for mice (see Figure 1A)
  2. Water bottle cap with sipper tube made in stainless steel (from now called Odor holder, see Figure 1B) (Tecniplast)
  3. Filter paper (Thermo Fisher Scientific, see Figure 1C and Figure 2)
  4. Opaque eppendorfs (1 ml volume) (Eppendorf, see Figure 1C)
  5. Scissors
  6. 10 µl pipet
  7. 10 µl pipet tips
  8. Tissue paper
  9. Standard table to place the test cage
  10. CCTV camera for an aerial view of the test
  11. Standard computer (PC or Mac)

    Figure 1. Basic material to perform the olfactory habituation test. A) Test cage made in plexiglass (12 cm width x 30 cm length x 13 cm height), with a circular perforation (5 cm diameter) in one of the extremes. B) Odor holder made in stainless steel (5 cm height from the base to the top of the sipper tube; base of 5 cm diameter; the hole of the sipper tube of 3 mm diameter). C) Standard opaque Eppendorf (left) containing the odor solution and the filter paper (3 cm height x 1 cm width) used to administer the odor. This filter paper is introduced in the sipper tube of B).


  1. Video recording software (GrabBee, grabbee.software.informer.com)
  2. Software to analyze exploratory behavior (Behav_Scor_v3.0_beta)


  1. Test cage
    1. A circular perforation (5 cm diameter) is made in the floor in one of the extremes of the plexiglass cage (see Figure 1A). This modification allows the odor holder to be placed between the table and the test cage, keeping it stable and resistant to any pressure made by the mouse.
  2. Preparation of the odor solution
    1. A stock solution is prepared by adding 1 µl of the odor, either almond or banana, to 1 ml of mineral oil (0.1% concentration) which is then aliqouted into 50 µl lots/batches in Eppendorf tube.
    2. All the tubes were sealed using parafilm to prevent evaporation from the tubes and were then stored at room temperature in a cardboard box protected from the light.
  3. Preparation of the scented filter paper
    1. The filter paper is cut into small pieces (1 cm x 3 cm, Figure 1C).
    2. For the olfactory habituation test, 5 µl of the diluted odorant (0.1%) or mineral oil is applied using a pipet to one edge of the pre-cut filter paper, which is then placed inside of the odor holder, introducing only two thirds of the scented filter paper (see Figure 2).
      Note: It is important not to introduce completely the filter paper in the holder in order to avoid physical contact (i.e. with the tongue) from the mouse. When this happens there is high probability that the mouse will bite the odor source, making it difficult to score the olfactory exploration.
    3. Three odor holders were used for the test, one dedicated to the mineral oil, another to the banana odor and the last one to the almond odor. The holders were cleaned with 20% ethanol after every use.

    Figure 2. Procedure to place the scented filter paper inside the odor holder. The filter paper is bent along its longitudinal axis, and is then introduced (two thirds) into the tube of the odor holder. Before placing the odor holder into the test cage, verify that the filter paper does not change position.

  4. Olfactory habituation test
    1. The test is performed during the light cycle with a standard temperature in an experimental room separate from the housing room.
    2. On day 0 the animals are food deprived for 24 h, starting 4 h after the onset of the light cycle.
    3. On day 1 (the test day) 30 min before the task all the necessary material (see above)  is prepared. The test cage is placed on the table and cleaned with 20% ethanol. Then, all the parameters concerning the video recording and scoring software are established, such as camera focus and contrast, position of the test cage in the right camera view field, etc.
    4. Once everything is set up, the first holder containing the mineral oil is placed in the test cage and the video recording is started. The mouse is placed in one of the extremes of the test cage opposite to the odor holder. The task consists of 5 min exposure to mineral oil or odor separated by 5 min inter-trial intervals; during this time the mouse remains in the test cage.
    5. In a given session, mice are first presented with only the holder containing the mineral oil. They are then presented with the first odorant during two successive trials (habituation).  Subsequently, they are presented the second odorant. The Figure 3 schematizes the different steps of the protocol.  
      Note: The presentation of the second odor is reflecting whether the animals can discriminate between odors, which are based on animal’s tendency to explore novel smell, and to control for a potential disinterest in the test odor.

    Figure 3. Olfactory habituation protocol. Immediately after arrival, mice are placed in individual cages for one week, where the body weight and food intake are monitored daily. On the test day, food deprived (24 h) animals are placed in the test cage and submitted to the presentation of first, the mineral oil, then, twice the odor A (almond or banana) and finally the odor B (almond or banana). Each presentation is followed by a rest time in which no odor or oil is presented. The time of exploration of the odor source is scored.

Representative data

  1. A representative habituation/ dishabituation curve is presented in Figure 4B. Exploration of the odor source was defined as directing the nose at a distance <1 cm from the tip of the holder, with the vibrissae moving, and/or touching it with the nose. The exploration was not scored when the mouse was chewing the object.
  2. The test cage was cleaned with ethanol 20% between the sessions. The two odorants used were presented in accordance with a counterbalanced schedule.

    Figure 4. Olfactory habituation/dishabituation in fasted mice. A) Odor source exploration by a fasted mouse. Exploration of the odor source was defined as directing the nose at a distance <1 cm from the tip of the holder, with the vibrissae moving and/or touching it with the nose. B) Representative habituation/dishabituation curve of a group of a 24 h fasted wild type mice. Note the decrease in the exploration (habituation) after the second presentation of a given odor (almond or banana). The exploration is recovered (dishabituation) after the presentation of a different odor (almond or banana).


  1. All animals are housed in individual cages for at least one week before the experiment.
  2. Bedding is changed at the start of food deprivation to prevent remnants of food from remaining in the cage
  3. This test is suitable to evaluate olfactory exploration in fasted mice (C57/BL6N). Non-fasted mice present low levels of exploration at the tested odorant concentration (0.1%).
  4. The olfactory habituation task was tested in several mice lines. We found more variability in the C57/BL6N mice than in the animals produced in our own institute. This could be due to a potential higher level of stress, associated with transport or housing conditions.


We would like to thank Samantha James and Annelot Van Esbroeck (Neurocentre Magendie) for the critical reading of this manuscript. This protocol was adapted according to McNamara et al. (2008), Fadool et al. (2004) and Ferguson et al. (2000). This work was supported by INSERM (G.M.), EU–Fp7 (REPROBESITY, HEALTH–F2–2008–223713, G.M.), European Research Council (ENDOFOOD, ERC–2010–StG–260515, G.M.), Fondation pour la Recherche Medicale (G.M.), Region Aquitaine (G.M.), LABEX BRAIN (ANR-10-LABX-43), Fyssen Foundation (E.S.–G.), We thank to Alexandre Desprez for its valuable technical assistance.


  1. Soria-Gomez, E., Bellocchio, L., Reguero, L., Lepousez, G., Martin, C., Bendahmane, M., Ruehle, S., Remmers, F., Desprez, T., Matias, I., Wiesner, T., Cannich, A., Nissant, A., Wadleigh, A., Pape, H. C., Chiarlone, A. P., Quarta, C., Verrier, D., Vincent, P., Massa, F., Lutz, B., Guzman, M., Gurden, H., Ferreira, G., Lledo, P. M., Grandes, P. and Marsicano, G. (2014). The endocannabinoid system controls food intake via olfactory processes. Nat Neurosci 17(3): 407-415.
  2. McNamara, A. M., Magidson, P. D., Linster, C., Wilson, D. A. and Cleland, T. A. (2008). Distinct neural mechanisms mediate olfactory memory formation at different timescales. Learn Mem 15(3): 117-125.
  3. Fadool, D. A., Tucker, K., Perkins, R., Fasciani, G., Thompson, R. N., Parsons, A. D., Overton, J. M., Koni, P. A., Flavell, R. A. and Kaczmarek, L. K. (2004). Kv1.3 channel gene-targeted deletion produces "Super-Smeller Mice" with altered glomeruli, interacting scaffolding proteins, and biophysics. Neuron 41(3): 389-404.
  4. 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.
  5. Linster, C., Menon, A. V., Singh, C. Y. and Wilson, D. A. (2009). Odor-specific habituation arises from interaction of afferent synaptic adaptation and intrinsic synaptic potentiation in olfactory cortex. Learn Mem 16(7): 452-459.
  6. Qiu, Q., Scott, A., Scheerer, H., Sapkota, N., Lee, D. K., Ma, L. and Yu, C. R. (2014). Automated analyses of innate olfactory behaviors in rodents. PLoS One 9(4): e93468.
  7. Slotnick, B. and Restrepo, D. (2005). Olfactometry with mice. Curr Protoc Neurosci: 8.20. 21-28.20. 24.
  8. Tan, J., Savigner, A., Ma, M. and Luo, M. (2010). Odor information processing by the olfactory bulb analyzed in gene-targeted mice. Neuron 65(6): 912-926.
  9. Yang, L., Zou, B., Xiong, X., Pascual, C., Xie, J., Malik, A., Xie, J., Sakurai, T. and Xie, X. S. (2013). Hypocretin/orexin neurons contribute to hippocampus-dependent social memory and synaptic plasticity in mice. J Neurosci 33(12): 5275-5284.
  10. Yang, M. and Crawley, J. N. (2009). Simple behavioral assessment of mouse olfaction. Curr Protoc Neurosci Chapter 8: Unit 8 24.


感觉知觉受到个体内部状态的严格调控。 特别地,已经显示嗅觉过程不断地受到反映身体的能量状态的代谢信号的影响。 因此,重要的是实施新的方法来评估身体能量变化对嗅觉性能的影响。 在这里,我们描述了一个行为协议,以准确地评估嗅觉习惯的禁食小鼠(索里亚 - 戈麦斯等人,2014)使用老鼠熟悉的基本设备。 简言之,将小鼠置于测试笼中,其中首先呈现无臭溶剂(对照),然后是气味A(两次),最后是气味B.该测试依赖于动物存在 在相同嗅觉刺激的几次展示后的行为反应。

关键字:食物的摄入量, 禁食的, 老鼠, 嗅觉的习惯, 嗅觉测试


  1. 动物:C57/BL6N雄性小鼠(Janvier Labs)
  2. 矿物油(Sigma-Aldrich,目录号:M-5904)
  3. 乙酸异戊酯(香蕉气味)(Sigma-Aldrich,目录号:W205508)
  4. 苯甲醛(杏仁气味)(Sigma-Aldrich,目录号:418099)
  5. 20%乙醇 注意:矿物油被用作溶剂,因为其文献中报道的无臭性质(Linster等人,2009; Qiu等人,2014; Slotnick和Restrepo,2005; Tan等人,2010 )。 杏仁和香蕉气味被用作 新颖,中性的气味(Yang等人,2013; Yang和Crawley,2009)。 然而,其他气味也适合在我们的条件下测试,如己醛。


  1. 用于小鼠的标准单个有机玻璃笼(见图1A)
  2. 水瓶盖与不锈钢制成的吸管(从现在称为气味支架,见图1B)(Tecniplast)
  3. 滤纸(Thermo Fisher Scientific,见图1C和图2)
  4. 不透明的eppendorfs(1ml体积)(Eppendorf,参见图1C)
  5. 剪刀
  6. 10μl移液器
  7. 10μl移液器吸头
  8. 纸巾
  9. 用于放置测试笼的标准表
  10. CCTV摄像机用于测试的鸟瞰图
  11. 标准计算机(PC或Mac)

    图1.进行嗅觉习惯测试的基本材料 A)用有机玻璃(12cm宽×30cm长×13cm高)制成的测试笼,其具有圆形穿孔(5cm直径)其中一个极端。 B)由不锈钢制成的气味保持器(从底部到吸管顶部5cm高;底部直径5cm;吸管管的孔直径3mm)。 C)含有气味溶液和用于施用气味的滤纸(3cm高×1cm宽)的标准不透明Eppendorf(左)。将该滤纸引入B)的吸管中


  1. 视频录制软件(GrabBee, grabbee.software.informer.com
  2. 分析探索行为的软件(Behav_Scor_v3.0_beta)


  1. 测试笼
    1. 在地板中的有机玻璃笼的一个极限中制造圆形穿孔(直径5cm)(参见图1A)。 这种修改允许气味保持器放置在桌子和测试笼之间,保持其稳定并且抵抗由鼠标产生的任何压力。
  2. 制备气味溶液
    1. 通过将1μl的气味(杏仁或香蕉)添加到1ml矿物油(0.1%浓度)中来制备储备溶液,然后将其在Eppendorf管中以50μl批次/批次混合。
    2. 使用石蜡膜密封所有的管,以防止从管中蒸发,然后在室温下储存在防护光的纸板箱中。
  3. 有香味的滤纸的制备
    1. 将滤纸切成小块(1cm×3cm,图1C)。
    2. 对于嗅觉习惯测试,使用移液管将5μl稀释的气味剂(0.1%)或矿物油施加到预先切割的滤纸的一个边缘,然后将其放置在气味保持器内部,仅引入三分之二香味滤纸(见图2)。
    3. 三个气味固定器用于测试,一个专用于矿物油,另一个用于香蕉气味,最后一种用于杏仁气味。每次使用后,用20%乙醇清洁支架。

    图2.将有香味的滤纸放置在气味固定器内的步骤。滤纸沿其纵向轴线弯曲,然后(三分之二)被引入气味固定器的管中。 在将气味固定器放入测试笼之前,请确认滤纸不会改变位置
  4. 嗅觉习惯测试
    1. 在光循环期间在与容纳室分开的实验室中使用标准温度执行测试。
    2. 在第0天,在光周期开始后4小时开始,将动物禁食24小时。
    3. 在第1天(测试日)任务前30分钟所有必要的材料(见上文) 。 将测试笼放置在台上并用20%乙醇清洁。 然后,所有的参数 涉及视频记录和记分软件,诸如相机聚焦和对比度,测试笼在右侧相机视野中的位置等。
    4. 一旦所有设置完成,包含矿物油的第一个支架放置在测试笼中,并开始视频录制。将小鼠放置在测试笼的与气味保持器相对的极端之一中。任务包括暴露于矿物油或气味5分钟间隔5分钟;在此期间,小鼠保持在测试笼中。
    5. 在给定的时间段中,首先仅给小鼠提供含有矿物油的持有者。然后在两个连续的试验(习惯性)期间,他们被提供第一种气味剂。随后,它们呈现为第二种气味剂。图3示意了协议的不同步骤。  



  1. 在图4B中给出了代表性的习惯/饮食习惯曲线。气味源的探测被定义为将鼻子指向距离保持器的尖端<1cm的距离,其中震动移动,和/或与鼻子接触。当鼠标咀嚼物体时,不进行探测。
  2. 在两次试验之间用20%乙醇清洗测试笼。使用的两种气味剂是按照平衡时间表

    图4.禁食小鼠中的嗅觉习惯/直立性。 A)通过禁食小鼠的气味源探测。气味源的探测被定义为将鼻子指向距离保持器的尖端<1cm的距离处,其中震动与鼻子移动和/或与鼻子接触。 B)一组24小时禁食野生型小鼠的代表性习惯/dishabituation曲线。注意在给定气味(杏仁或香蕉)的第二次呈现后,探测(习惯)的减少。在呈现不同的气味(杏仁或香蕉)之后,回收(dishabituation)。


  1. 所有动物在实验前在单笼中饲养至少一周。
  2. 在食物匮乏开始时更换床上用品以防止残留食物残留在笼子中
  3. 该试验适合于评价禁食小鼠(C57/BL6N)中的嗅觉探查。 非禁食小鼠在测试的气味浓度(0.1%)下呈现低水平的探索。
  4. 在几个小鼠系中测试嗅觉习惯任务。 我们发现在C57/BL6N小鼠比在我们自己的研究所生产的动物更多的变异性。 这可能是由于潜在的更高水平的压力,与运输或住房条件有关


我们要感谢Samantha James和Annelot Van Esbroeck(Neurocentre Magendie)对这份手稿的批判性阅读。该方案根据McNamara等人(2008),Fadool等人, (2004)和Ferguson (2000)。这项工作由INSERM(GM),EU-Fp7(REPROBESITY,HEALTH-F2-2008-223713,GM),欧洲研究理事会(ENDOFOOD,ERC-2010-StG- 260515,GM),Fondation pour la Recherche医疗GM),地区阿基坦(GM),LABEX BRAIN(ANR-10-LABX-43),Fyssen基金会(ES-G),我们感谢Alexandre Desprez的宝贵技术援助。


  1. Soria-Gomez,E.,Bellocchio,L.,Reguero,L.,Lepousez,G.,Martin,C.,Bendahmane,M.,Ruehle,S.,Remmers,F.,Desprez,T。,Matias,I 。,Wiesner,T.,Cannich,A.,Nissant,A.,Wadleigh,A.,Pape,HC,Chiarlone,AP,Quarta,C.,Verrier,D.,Vincent,P.,Massa, Lutz,B.,Guzman,M.,Gurden,H.,Ferreira,G.,Lledo,PM,Grandes,P.and Marsicano,G。(2014)。 内源性大麻素系统通过嗅觉过程控制食物摄取。

    Nat Neurosci < em> 17(3):407-415。
  2. McNamara,A.M.,Magidson,P.D.,Linster,C.,Wilson,D.A。和Cleland,T.A。(2008)。 不同的神经机制介导不同时间尺度的嗅觉记忆形成。 Learn Mem 15(3):117-125。
  3. Fadool,D.A.,Tucker,K.,Perkins,R.,Fasciani,G.,Thompson,R.N.,Parsons,A.D.,Overton,J.M.,Koni,P.A.,Flavell,R.A。和Kaczmarek, Kv1.3通道基因定位缺失产生具有改变的肾小球的"超级嗅探小鼠",相互作用的支架蛋白质和生物物理学。 Neuron 41(3):389-404。
  4. Ferguson,J.N.,Young,L.J.,Hearn,E.F.,Matzuk,M.M.,Insel,T.R.and Winslow,J.T。(2000)。 缺乏催产素基因的小鼠的社会健忘症。 > 25(3):284-288。
  5. Linster,C.,Menon,A.V.,Singh,C.Y。和Wilson,D.A。(2009)。 气味特异性习惯起因于传入突触适应和嗅皮质中内在突触增强的相互作用。 a> Learn Mem 16(7):452-459。
  6. Qiu,Q.,Scott,A.,Scheerer,H.,Sapkota,N.,Lee,D.K.,Ma,L.and Yu,C.R。(2014)。 自动分析啮齿类动物的先天嗅觉行为 PLoS One 9(4):e93468。
  7. Slotnick,B。和Restrepo,D。(2005)。 使用鼠标的嗅觉测量。 > Curr Protoc Neurosci :8.20。 21-28.20。 24.
  8. Tan,J.,Savigner,A.,Ma,M。和Luo,M。(2010)。 在基因靶向小鼠中分析的嗅球的气味信息处理。 Neuron 65(6):912-926。
  9. Yang,L.,Zou,B.,Xiong,X.,Pascual,C.,Xie,J.,Malik,A.,Xie,J.,Sakurai,T。和Xie, hypocretin/orexin神经元有助于小鼠中海马依赖的社交记忆和突触可塑性。 J Neurosci 33(12):5275-5284。
  10. Yang,M。和Crawley,J.N。(2009)。 对小鼠嗅觉的简单行为评估。 Curr Protoc Neurosci 第8章:单元8 24.
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引用:Desprez, T., Marsicano, G. and Soria-Gómez, E. (2014). Olfactory Habituation in Fasted Mice. Bio-protocol 4(20): e1267. DOI: 10.21769/BioProtoc.1267.