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Assessment of Olfactory Behavior in Mice: Odorant Detection and Habituation-Dishabituation Tests
小鼠嗅觉行为的评估;气味检测以及习惯化与去习惯化测试   

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Neuron
Aug 2014

Abstract

Olfaction has adaptive value for rodents as it is essential for feeding and mating, the establishment of social and territorial relationships, or the detection of potential predators, among others (Apfelbach et al., 2005). Sensory input from the olfactory mucosa is first processed in the main olfactory bulb (MOB), a telencephalic structure that exhibits neurogenesis during the lifespan of the animal. Changes in MOB circuitry due to neuronal dysfunction or changes in interneuron turnover rate affect olfactory performance in different ways (Fleming et al., 2008; Breton-Provencher et al., 2009; Attems et al., 2014). Alterations in adult MOB neurogenesis, in particular, result in changes in odorant discrimination which can be assayed in habituation-dishabituation behavioral paradigms (Mouret et al., 2009; Delgado et al., 2014). Here, we present a simple protocol for the quantitative assessment of two olfactory tasks that can be used to detect neurogenic alterations in the MOB (Delgado et al., 2014). The procedure has been optimized to require little time and can, therefore, be used to analyze genetically modified mice that are housed in an isolated specific pathogen-free (SPF) mouse facility.

Materials and Reagents

  1. Animals
    Laboratory-bred experimentally-naïve male mice. Both CD1 and C57BL/6J strains have been successfully tested. Male mice are housed in groups of 4-5 per cage, kept in an environment with controlled temperature (23 ± 2 °C) and humidity under a 12-12 h light-dark cycle with food and water ad libitum. The use of only one gender reduces inter-individual variation. Males are used instead of females to avoid cycling hormone effects associated to oestrus (Jemiolo et al., 1986).
    Note: All mice must be of equal or similar age (i.e. always less than two weeks apart, as the rate of neurogenesis is age-dependent). When this is not possible, cohorts of similar sizes for each experimental condition can be tested separately at different times, but this schedule results in greater variability and will, therefore, require a higher total number of animals. In most of our experiments a minimum of 7 mice were required to reach statistical significance. However, 10-12 mice per group are recommended to minimize the effects of inter-individual variation.

  2. Odorants
    Odorants must have non-emotional value. We use two synthetic odorants: citralva (geranonitrile, 3,7-dimethyl-2,6-octadien-1-nitrile), a lemon-like citrus smell, and geraniol (3,7-dimethyl-2,6-octadien-1-ol), a sweet rose-like scent, at dilutions that range between 1:160 to 1:10. Both odorants have been tested before in olfactory experiments (Luo et al., 2002; Delgado et al., 2014) although others, i.e. banana odor (isoamyl acetate), almond odor (benzaldehyde), or hexanal, have also been used in similar tests (see, for example, Mandairon et al., 2006; Fleming et al., 2008). Odorants can be obtained from different suppliers, such as Ventós S.A (dealer of International Flavors and Fragrances Inc. Barcelona, Spain). Because synthetic fragrances are usually available in an oleic format, we use mineral oil for their dilution. It is important to keep them always in tightly sealed containers to minimize their evaporation.

  3. Other materials
    1. Mineral oil (Sigma-Aldrich, catalog number: M5904 )

Equipment

Olfactory experiments are performed in a dedicated lab room with positive pressure (10-15 gauges) and frequent air reposition (no less than 20 times per hour) for fast removal of odors. Indirect dim lighting is recommended for testing. Essence dilutions should be prepared in a different, not connected room.
The materials used (see Figure 1) are:

  1. A 22.5 x 22.5 x 29.5 cm open Plexiglas box with solid light green walls and a hole of 1 cm of diameter in one of the sides, located at 8 cm above the box ground and at 11.2 cm from the lateral corners of the box. A transparent methyl methacrylate solid cover is advisable to avoid essence dissipation during the tests. The video recording system is fixed above the exploration box at a distance of 30-40 cm from the transparent cover in order to video-tape at good resolution without interfering with the test.
    Note: The olfactory exploration box can be custom-made following the description. The box is thoroughly wiped clean with 10% alcohol and dried before and after the experiment, as well as between animals. After each test, the used soiled bedding has to be carefully removed with a household vacuum cleaner and replaced by fresh bedding. Use odorless disposable paper towels and nitrile gloves for cleaning and drying.
  2. Regular cotton sticks bought in any drugstore
  3. Lab timer
  4. Micropipette (2-20 or 20-200 µl-range) and appropriate disposable tips
  5. Adhesive tape
  6. 1.5 ml microcentrifuge tubes to preserve the essences and a microcentrifuge tube rack to avoid fluid-containing tubes from tipping over
  7. A sealed container (optional if the garbage bag is sealed) can be used for disposal of any objects that are impregnated with essences.

Procedure

  1. Threshold detection
    This protocol is designed to measure odorant sensitivity by determining the lowest concentration of a certain odor that is perceivable by the sense of smell.
    The group of mice that is going to be tested is kept in ventilated racks in a different room. Each mouse is extracted from its cage under a hood and placed in an empty clean box for transportation to the testing room. After the test, the mouse is returned to the original cage and the transportation box is thoroughly wiped clean.
    1. General habituation to the testing conditions
      The mice are first exposed to the general environment and procedure.
      1. The mouse is gently placed in the testing box (Figure 2) previously covered with a uniform layer of clean bedding not exceeding 1 cm in height. We recommend covering the box with the transparent lid during testing.
        Note: An excess of bedding in the testing box stimulates exploratory behaviors, such as scratching and digging, which may distract the mouse from the main task. On the contrary, mouse mobility is hindered and interferences with depositions are increased if the bedding is too little or absent at all.
      2. The mouse is allowed a 3-min period of free exploration for context processing and habituation (Fanselow, 2000).
      3. A cotton stick soaked in mineral oil is introduced into the box through the little hole located in one of the box walls. The stick should protrude from the wall approximately 3 cm and it is held in place for 1 min.
        Note: To impregnate the sticks, take 20 µl of the solution with a micropipette, close the tube lid immediately afterwards and deposit the 20 µl-drop directly into the swab head. An easy way to hold the stick in place without vibrations during the one-minute exposure is to use sticks that have been manually bent at right angles. Once the soaked end of the stick is passed through the hole, the other end can be easily fixed to the outside of the box wall with adhesive tape. It takes only a few seconds to change the stick. Avoid touching the testing box walls or the inner part of the hole with the impregnated swab. Use a lab timer without acoustic alarm to control time.
      4. The first exposure to the mineral oil-soaked stick is followed by 4 more successive exposures to new sticks freshly soaked in the same mineral oil to produce a habituation to the stick and to its movement in and out of the box. This part of the test can also reveal differences between genotypes/strains in their exploratory response to novel objects.
      5. Use a digital video system to record the behavior of the animal throughout the testing.
    2. Threshold detection
      After the 5 trials with the non-odorant stimulus, the mouse will be exposed to cotton sticks with increasing concentrations of one of the scents (diluted in mineral oil at 1:160, 1:80, 1:40, 1:20, and 1:10) to determine the minimum concentration that triggers exploration by the animal. We usually employ citralva for this task, but it can be done with any of the two odorants.
      1. A cotton stick soaked in the scent at the lowest concentration is introduced into the box, as done previously for sticks with mineral oil, and held in place for 1 min.
        Note: Use a new stick for every trial and immediately discard used sticks by placing them in a sealed container or zipped garbage bag.
      2. A cotton stick soaked in mineral oil is then used in the same way.
      3. Next stick is soaked in the same scent but at double the concentration of the previous one.
      4. The procedure is then repeated until all concentrations have been tested.
      5. Use a digital video system to record the behavior of the animal throughout the testing.
        The inter-trial interval (ITI) is defined as the time between exposures to the odorants. This interval should allow the mouse to show specific olfactory behaviors towards each scented stick without resulting in a general loss of interest in the task. For this test, an ITI of 1-2 min is recommended. Indeed, we use a one-minute ITI, during which we expose the animal to a stick impregnated in mineral oil (see step A2g). It is important to keep in mind that a very prolonged ITI can change the novelty value of each trial (Breton-Provencher et al., 2009) whereas very short ITIs can induce one habituation to context too fast (Sanderson and Bannerman, 2011).
        Differences in the behavioral performance of mice in the control and experimental groups under analysis in this test indicate differences in olfactory perception (see, for example, Delgado et al., 2014; Figure 3A).

  2. Olfactory habituation-dishabituation
    The animals will be exposed to two different scents to test olfactory discrimination, or how the animals perceive the difference. This protocol is designed to test the habituation to a specific odor, indicated by the progressive decrease in the exploration time in successive presentations, and the dishabituation, indicated by increased exploration of a novel, distinct odor.
    This second test should be run with a set of mice that is different from the ones tested in the odorant detection test, as novel and familiar odors are coded differently (Kato et al., 2012) and familiarity of odors could influence olfactory exploration and the grade of stimulus novelty perceived by the mouse.
    1. General habituation to the testing conditions (follow section 1 in Procedure A).
    2. Habituation/dishabituation: After the 5 trials with the non-odorant stimulus, the mouse will be exposed to 5 presentations of one scent followed by 5 exposures to a different scent.
      Note: A concentration of odorant that is perceived equally well by the control and experimental group will be used for this task. We routinely use 1:20.
      1. A cotton stick impregnated with 20 µl of geraniol at 1:20 is introduced into the box, as done previously for sticks with mineral oil, and held in place for 1 min.
        Note: Use a new stick for every trial and immediately discard used sticks by placing them in a sealed container or zipped garbage bag.
      2. A cotton stick soaked in mineral oil is then used in the same way.
      3. Steps B2a-b are repeated 4 more times.
      4. A cotton stick impregnated with 20 µl of citralva at 1:20 is introduced into the box and held in place for 1 min.
      5. A cotton stick soaked in mineral oil is then used in the same way.
      6. Steps B2d-e are repeated 4 more times.
      7. Use a digital video system to record the behavior of the animal throughout the testing.
        Habituation is reflected as a reduction in exploratory time after the first or first and second exposures to the same odor; dishabituation is reflected as a renovated interest in the novel odorant stimulus (see Figure 3B).
        It is important to use a scent concentration that is appropriate for the test. The same odorant molecule can induce attractive or repulsive responses depending on its concentration (Yoshida et al., 2012). Moreover, olfactory sensory receptors may fail to generate action potentials at very high odorant concentrations (Ghatpande and Reisert, 2011). We recommend using the lowest concentration at which all animals clearly perceived the olfactory stimulus in the threshold detection test; that is, the concentration at which more animals exhibit the highest exploration time. In our experiments with geraniol and citralva in CD1 and C57BL/6J mice, these concentrations were either 1:40 or 1:20.

Data analysis

In both types of experiment, the total time spent by each mouse performing any type of olfactory exploration action is assessed for every trial in the video recordings after the experiment is finished (Figure 2B and Video 1). Actions evaluated as olfactory exploration include touching the cotton or smelling, sniffing or heading the nose towards the stick at a close distance. The Smart Junior® software (Panlab, Spain) can be used to track all behaviors and to measure the time each mouse spends exploring the stick. The measurements can also be done manually using the video recording. The variables to compare statistically are “exploration time” and “trial” as detailed in Figure 3.
For the statistical analyses, a mixed-model ANOVA of repeated measures (“trial” factor) is used, followed by post hoc comparisons (e.g. Tukey or Bonferroni tests) to determine the occurrence of habituation and dishabituation. Finally, a “between-subjects” factor can be added to the ANOVA for comparison of two experimental groups. Statistical differences (p ˂ 0.05) are represented by *(dishabituation) and # (habituation).


Figure 1. Equipment and materials. Photograph of the testing box and some of the required materials listed in the same order as they appear in the text. Arrow head points to the hole through which the scented stick is introduced into the exploratory box.


Figure 2. Evaluation scene. Illustrative image of a mouse during the test. Actions evaluated as olfactory exploration include touching the cotton or smelling, sniffing or heading the nose towards the stick at a close distance. For this, the image of a square with a side length of 6 cm centered at the swab tip is overlapped by the software in the video image. Computer-assisted evaluation may not be necessary if spatial references are set-up manually by the observer and kept standardized across sessions.


Figure 3. Representative results. A. Threshold detection test. Histogram showing the mean exploration time (in seconds) ± s.e.m. of a number of mice in trials with different concentrations of citralva interspersed by mineral oil sticks. For the analysis, a Student´s t-test comparing the time of exploration of the odorant stick vs. the non-odorant stick at each concentration can be used to analyze differences in odor detection. Threshold detection is established as the minimum scent concentration at which a significant difference is found (1:160 in the example). Thresholds for control and experimental groups can be compared (see Delgado et al., 2014). B. Habituation-dishabituation test. Graph showing the mean exploration time (in seconds) ± s.e.m. of a number of mice in successive trials of mineral oil, geraniol (1:20), and citralva (1:20) in the indicated order. Habituation is reflected as less time sniffing successive same-odor trials. Habituation is generally accelerated when mice get familiarized with the task and, therefore, it occurs more rapidly during the testing of a second odorant. Dishabituation is reflected as more time sniffing a new smell and is analyzed by comparing “trial 5” with “new-smell trial 1”. Eventually, a rise in olfactory exploration in trial 1 and 2, or even trial 3 may occur, usually during exposure of naïve freely exploring mice to the first odorant (see geraniol vs. citralva responses).

Video 1. Example of recording. The video recording shows a normal two-month old CD1 male mouse exposed to: 1) a stick soaked in a dilution of citralva that is not detected by the animal (1:160), 2) a stick soaked in non-odorant mineral oil during the inter-trial interval, and 3) a stick soaked in a dilution of citralva that is detected by the mouse (1:80). Interest of the mouse towards the stick and olfactory responsive behavior can be observed in the recording of the third, but not the previous sticks. The video is a fragment of the recording of a real olfactory threshold experiment.

Acknowledgments

We thank the Servicios Centrales de Soporte a la Investigación Experimental and I. Noguera for technical assistance and animal care. I.F is supported by Fundación Botín and by Banco Santander through its Santander Universities Global Division, and by grants from Generalitat Valenciana (Programa Prometeo 2013/020and ISIC) and Ministerio de Economía y Competitividad (SAF2011-13332, CIBERNED CB06/05/0086, and RETIC TerCel RD12/0019/0008). In memoriam of our beloved friend Nicholas J. Mackintosh, Emeritus Professor of Experimental Psychology (University of Cambridge).

References

  1. Apfelbach, R., Blanchard, C. D., Blanchard, R. J., Hayes, R. A. and McGregor, I. S. (2005). The effects of predator odors in mammalian prey species: a review of field and laboratory studies. Neurosci Biobehav Rev 29(8): 1123-1144.
  2. Attems, J., Walker, L. and Jellinger, K. A. (2014). Olfactory bulb involvement in neurodegenerative diseases. Acta Neuropathol 127(4): 459-475.
  3. Breton-Provencher, V., Lemasson, M., Peralta, M. R., 3rd and Saghatelyan, A. (2009). Interneurons produced in adulthood are required for the normal functioning of the olfactory bulb network and for the execution of selected olfactory behaviors. J Neurosci 29(48): 15245-15257.
  4. Delgado, A. C., Ferron, S. R., Vicente, D., Porlan, E., Perez-Villalba, A., Trujillo, C. M., D'Ocon, P. and Farinas, I. (2014). Endothelial NT-3 delivered by vasculature and CSF promotes quiescence of subependymal neural stem cells through nitric oxide induction. Neuron 83(3): 572-585.
  5. Fanselow, M. S. (2000). Contextual fear, gestalt memories, and the hippocampus. Behav Brain Res 110(1-2): 73-81.
  6. Fleming, S. M., Tetreault, N. A., Mulligan, C. K., Hutson, C. B., Masliah, E. and Chesselet, M. F. (2008). Olfactory deficits in mice overexpressing human wildtype alpha-synuclein. Eur J Neurosci 28(2): 247-256.
  7. Ghatpande, A. S. and Reisert, J. (2011). Olfactory receptor neuron responses coding for rapid odour sampling. J Physiol 589(Pt 9): 2261-2273.
  8. Jemiolo, B., Harvey, S. and Novotny, M. (1986). Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents. Proc Natl Acad Sci U S A 83(12): 4576-4579.
  9. Kato, H. K., Chu, M. W., Isaacson, J. S. and Komiyama, T. (2012). Dynamic sensory representations in the olfactory bulb: modulation by wakefulness and experience. Neuron 76(5): 962-975.
  10. 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 alpha subunit of G(o). Brain Res 941(1-2): 62-71.
  11. Mandairon, N., Sultan, S., Rey, N., Kermen, F., Moreno, M., Busto, G., Farget, V., Messaoudi, B., Thevenet, M. and Didier, A. (2009). A computer-assisted odorized hole-board for testing olfactory perception in mice. J Neurosci Methods 180(2): 296-303.
  12. Mouret, A., Lepousez, G., Gras, J., Gabellec, M. M. and Lledo, P. M. (2009). Turnover of newborn olfactory bulb neurons optimizes olfaction. J Neurosci 29(39): 12302-12314.
  13. Sanderson, D. J. and Bannerman, D. M. (2011). Competitive short-term and long-term memory processes in spatial habituation. J Exp Psychol Anim Behav Process 37(2): 189-199.
  14. Yoshida, K., Hirotsu, T., Tagawa, T., Oda, S., Wakabayashi, T., Iino, Y. and Ishihara, T. (2012). Odour concentration-dependent olfactory preference change in C. elegans. Nat Commun 3: 739.

简介

嗅觉对于啮齿动物具有适应性价值,因为它对于喂养和交配,建立社会和领域关系或检测潜在的捕食者是至关重要的(Apfelbach等人,2005)。来自嗅粘膜的感觉输入首先在主嗅球(MOB)中处理,该嗅球是在动物的寿命期间展示神经发生的端脑结构。由于神经元功能障碍或中间神经元转换率的变化,MOB电路的变化以不同的方式影响嗅觉性能(Fleming等人,2008; Breton-Provencher等人,2009年) ; Attems等人,,2014)。成人MOB神经发生的改变,特别是导致气味辨别的变化,其可以在习惯 - 习惯行为模式中进行测定(Mouret等人,2009; Delgado等人 >,2014)。在这里,我们提出了一个简单的协议,用于定量评估两个嗅觉任务,可用于检测MOB中的神经源性改变(Delgado等人,2014)。该方法已经被优化以需要很少的时间,并且因此可以用于分析容纳在分离的特异性无病原体(SPF)小鼠设备中的遗传修饰的小鼠。

材料和试剂

  1. 动物
    实验室培育的实验首次实验的雄性小鼠。 CD1和C57BL/6J菌株都已成功测试。将雄性小鼠以每笼4-5只的组饲养,保持在具有控制温度(23±2℃)和湿度的环境中,在12-12小时光 - 暗循环下,食物和水随意地em>。仅使用一种性别可以减少个体间的差异。使用男性而不是女性,以避免与发情相关的循环激素作用(Jemiolo等人,1986)。
    注意:所有小鼠必须具有相等或相似的年龄(即总是相隔不到两个星期,因为神经发生的速率是年龄依赖性的)。当这不可能时,可以在不同时间单独测试每个实验条件的相似大小的群组,但是该计划导致更大的变异性,并且因此将需要更高的动物总数。在我们的大多数实验中,需要最少7只小鼠达到统计学显着性。但是,建议每组10-12只小鼠,以尽量减少个体间差异的影响。

  2. 气味
    加味剂必须有非感情价值。我们使用两种合成添味剂:柠檬醛(香叶腈,3,7-二甲基-2,6-辛二烯-1-腈),柠檬样柑橘气味和香叶醇(3,7-二甲基-2,6-辛二烯-1 -ol),甜玫瑰样气味,稀释度为1:160至1:10。这两种气味剂在嗅觉实验中都已经过测试(Luo等人,2002; Delgado等人,2014),尽管其他人,例如香蕉气味(乙酸异戊酯),杏仁气味(苯甲醛)或己醛也已经用于类似的测试中(参见例如Mandairon等人,2006; Fleming等人,/em,2008)。气味剂可以从不同的供应商获得,例如VentósS.A(经销商 International Flavors and Fragrances Inc. Barcelona,Spain)。 因为合成香料通常以油酸形式提供,我们使用矿物油稀释。 将它们保存在密封的容器中以尽量减少其蒸发是非常重要的
  3. 其他材料
    1.矿物油(Sigma-Aldrich,目录号:M5904)

设备

在具有正压力(10-15计)和频繁空气重新定位(每小时不小于20次)的专用实验室中进行嗅觉实验以快速除去气味。 建议使用间接暗淡照明进行测试。 精华稀释液应在不同的连接室中准备。
使用的材料(见图1)是:

  1. 一个22.5×22.5×29.5厘米开口的有机玻璃盒,其具有实心浅绿色的壁和在其中一个侧面中的一个直径为1cm的孔,位于盒地面上方8cm处以及距离盒的侧向角11.2cm处。建议在测试期间使用透明的甲基丙烯酸甲酯固体覆盖物以避免散逸。视频记录系统固定在探测箱上方距离透明盖30-40厘米的距离,以便在不干扰测试的情况下以良好的分辨率录像。
    注意:嗅觉探查框可以根据描述进行定制。在实验之前和之后,以及在动物之间,用10%酒精彻底擦拭盒子并干燥。每次测试后,使用的污染床上用品必须用家用真空吸尘器小心取出,并换上新鲜的床上用品。使用无臭的一次性纸巾和丁腈手套清洁和干燥。
  2. 在任何药店买的普通棉棒
  3. 实验室计时器
  4. 微量移液器(2-20或20-200μl范围)和适当的一次性提示
  5. 胶带
  6. 1.5 ml微量离心管保存精华液和微量离心管架,以避免含有液体的试管倾倒
  7. 密封容器(如果垃圾袋密封,则可选)可用于处理浸渍有精华液的任何物体。

程序

  1. 阈值检测
    该协议旨在通过确定气味感觉到的某种气味的最低浓度来测量气味敏感性。
    将要测试的小鼠组保存在不同房间中的通风架中。将每只小鼠在其罩下从其笼中提取,并置于空的干净箱中以运送至测试室。试验后,将鼠标放回原来的笼子里,并将运输箱彻底擦拭干净
    1. 一般习惯适应测试条件
      小鼠首先暴露于一般环境和程序
      1. 将小鼠先前轻轻地放置在测试盒(图2)中 覆盖着不超过1厘米的干净床上的均匀层 高度。 我们建议在此期间用透明盖盖住盒子 测试 注意:测试盒中的床上用品过多刺激 探索性行为,如抓地和挖掘,这可能 分心鼠标从主要任务。 相反,小鼠的移动性 并且增加了沉积物的干扰 床铺太少或根本没有。
      2. 允许小鼠进行3分钟的自由探索以进行情境处理和习惯化(Fanselow,2000)
      3. 将浸泡在矿物油中的棉棒放入箱中 通过位于箱壁之一的小洞。 棍子 应该从墙壁突出约3厘米,并保持在适当的位置 1分钟。
        注意:要浸渍棒,取20μl的 溶液用微量移液管,然后立即关闭管盖 并将20μl滴直接放入拭子头。一个简单的方法 在一分钟内将棒保持在原位而没有振动 暴露是使用已经手动弯曲成直角的棒。 一旦棒的浸泡端通过孔,另一个 端部可以容易地用粘合剂固定到箱壁的外侧 胶带。只需要几秒钟更换棒。避免触摸 测试盒壁或孔的内部浸渍  棉签。使用无声音警报的实验室计时器控制时间。
      4. 第一次暴露于矿物油浸泡的棒之后是另外4个  连续暴露于新鲜浸泡在相同矿物中的新鲜棒 油产生习惯的棒和它的运动进出  的盒子。这部分测试也可以揭示之间的差异 基因型/菌株在其对新物体的探索响应。
      5. 使用数字视频系统记录动物在整个测试过程中的行为。
    2. 阈值检测
      在用非气味刺激的5次试验后,小鼠将是 暴露于棉花棒与增加浓度的一个 香味(在矿物油中以1:160,1:80,1:40,1:20和1:10稀释)至 确定触发探索的最小浓度 动物。 我们通常使用citralva完成这项任务,但可以做到 与任何两种气味剂
      1. 一根棉棒浸泡在 将最低浓度的香味引入盒子中,如所做的那样 先前用于矿物油棒,并保持1分钟 注意:对于每个试验使用一个新棒,并立即丢弃使用 请将它们放在密封容器或拉链垃圾袋中。
      2. 然后以相同的方式使用浸泡在矿物油中的棉棒。
      3. 下一根棍子浸泡在同一种香味中,但浓度是前一种香味的两倍
      4. 然后重复该程序,直到测试了所有浓度
      5. 使用数字视频系统记录动物在整个测试过程中的行为。
        试验间隔(ITI)定义为暴露之间的时间   到气味剂。 这个间隔应该允许鼠标显示具体 嗅觉行为朝向每个香味棒而不导致 一般损失的任务的兴趣。 对于该测试,ITI为1-2分钟 建议。 事实上,我们使用一分钟的ITI,在此期间我们公开 将动物给予浸渍在矿物油中的棒(参见步骤A2g)。 它是 重要的是要记住,非常长的ITI可以改变 每个试验的新颖性值(Breton-Provencher等人,2009),而 非常短的ITI可以诱导一个习惯上下文太快 (Sanderson和Bannerman,2011)。
        行为的差异 性能的对照组和实验组的小鼠 在该试验中的分析表明嗅觉感知的差异(参见,  例如,Delgado等人,2014;图3A)。

  2. 嗅觉习惯 - 习惯性
    动物将暴露于两种不同的香味以测试嗅觉辨别,或动物如何感知差异。该协议旨在测试对特定气味的适应性,通过连续展示中的探索时间的逐渐减少来表示,以及通过增加探索一种新颖的,独特的气味来指示的习惯性。 第二次测试应该用一组不同于气味检测试验中测试的小鼠进行,因为新的和熟悉的气味被不同地编码(Kato等人,2012)和熟悉气味可以 影响嗅觉探索和小鼠感知的刺激新颖程度
    1. 一般习惯于测试条件(按照程序A的第1节)
    2. 习惯/dishabituation:5次试验后用非气味剂 刺激,小鼠将暴露于一种气味的5个呈现 然后5次暴露于不同的香味 注意:浓度 的气味,其被对照感觉到同样良好 实验组将用于此任务。 我们经常使用1:20。
      1. 以1:20浸渍20μl香叶醇的棉棒 引入盒中,如前面对于具有矿物油的棒所做的,   并保持在适当位置1分钟。
        注意:对于每个试用,请使用新的棒   并立即丢弃用过的棒通过将它们放在密封 集装箱或拉链垃圾袋。
      2. 然后以相同的方式使用浸在矿物油中的棉棒
      3. 步骤B2a-b重复4次以上。
      4. 将以1:20浸渍20μl柠檬醛的棉棒引入盒中并保持在适当位置1分钟。
      5. 然后以相同的方式使用浸在矿物油中的棉棒
      6. 步骤B2d-e重复4次以上。
      7. 使用数字视频系统记录动物在整个测试过程中的行为。
        习惯反映为探索时间之后的减少 第一次或第一次和第二次暴露于相同的气味; 盘居式是 反映为新颖的气味刺激的重新兴趣(见 图3B)。
        使用气味浓度是很重要的 适合测试。 相同的气味分子可以诱导 吸引力或排斥反应取决于其浓度 (Yoshida等人,2012)。 此外,嗅觉感觉受体可能失败 以在非常高的加臭剂浓度下产生动作电位 (Ghatpande和Reisert,2011)。 我们建议使用最低 所有动物清楚地感觉到嗅觉的浓度 刺激在阈值检测测试; 即,浓度 其中更多的动物展示最高的勘探时间。 在我们的 实验与香叶醇和citralva在CD1和C57BL/6J小鼠,这些 浓度为1:40或1:20。

数据分析

在两种类型的实验中,在实验完成之后,对每个进行任何类型的嗅觉探测动作的小鼠所进行的每次试验在视频记录中进行评估(图2B和视频1)。被评价为嗅觉探索的行为包括接触棉花或嗅闻,嗅探或朝着棍子靠近的方向前进。 Smart Junior ®软件(Panlab,西班牙)可用于跟踪所有行为并测量每只老鼠花费在探索棒上的时间。测量也可以使用视频记录手动完成。统计比较的变量是"探索时间"和"试验",如图3所示。
对于统计分析,使用重复测量的混合模型ANOVA("试验"因子),随后进行事后比较(例如Tukey或Bonferroni测试)以确定习惯和习惯性的发生。最后,可以将"受试者之间"因子添加到ANOVA中以用于两个实验组的比较。统计学差异(p = 0.05)用*(dishabituation)和#(习惯性)表示

图1.设备和材料。测试框的照片以及列在文本中的相同顺序列出的一些必需材料。箭头指向通过其将香棒引入探测盒的孔。


图2.评估场景。测试期间鼠标的说明图像。评估为嗅觉探索的行为包括接触棉花或嗅到,嗅探或朝向棍子向鼻子靠近 距离。为此,以拭子尖端为中心的边长为6cm的正方形的图像与视频图像中的软件重叠。如果空间参考由观察者手动建立并在会话之间保持标准化,则可能不需要计算机辅助评估。


图3.代表结果 A.阈值检测测试。直方图显示平均探测时间(秒)±s.e.m。的许多小鼠在不同浓度的柠檬醛穿插在矿物油棒的试验。对于分析,将在每个浓度下比较气味棒的探测时间与非气味棒的时间的学生t检验可用于分析气味检测的差异。阈值检测被建立为找到显着差异的最小气味浓度(在该实例中为1:160)。可以比较对照组和实验组的阈值(参见Delgado等人,2014年)。 B.习惯 - 习惯测试。图表显示平均勘探时间(秒)±s.e.m。的矿物油,香叶醇(1:20)和柠檬醛(1:20)以指定顺序进行的连续试验中的许多小鼠。习惯反映为较少的时间嗅闻连续相同气味的试验。当小鼠熟悉该任务时,习惯通常加速,因此,在测试第二种气味剂期间它更快地发生。习惯性反映为更多的时间嗅新气味,并通过比较"试验5"与"新气味试验1"进行分析。最终,试验1和2,甚至试验3中的嗅觉探索的增加可能发生,通常在初始自由探索小鼠暴露于第一种气味剂(参见香叶醇与柠檬醛反应)期间。

视频1.录制示例。视频录制显示正常的两个月大的CD1雄性小鼠暴露于:1)浸泡在柠檬醛稀释液中的棒,动物未检测到(1: 160),2)在试验间隔期间浸泡在非气味矿物油中的棒,和3)浸泡在由小鼠检测的柠檬醛稀释物(1:80)中的棒。鼠标对棒的兴趣和嗅觉响应行为可以在第三个记录中观察到,但不是先前的棒。视频是真实的嗅觉阈值实验的记录的片段。

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致谢

我们感谢Servicios Centrales de Soporte a laInvestigaciónExperimental和I. Noguera的技术援助和动物护理。 IF由FundaciónBotín和桑坦德银行通过其桑坦德大学全球分部以及来自瓦伦西亚大学(Programa Prometeo 2013/020and ISIC)和经济部长竞争奖学金(SAF2011-13332,CIBERNED CB06/05/0086)的资助, RETIC TerCel RD12/0019/0008)。在我们心爱的朋友尼古拉斯J.麦金托什,实验心理学荣誉教授(剑桥大学)的纪念。

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引用:Perez-Villalba, A., Palop, M. J., Pérez-Sánchez, F. and Fariñas, I. (2015). Assessment of Olfactory Behavior in Mice: Odorant Detection and Habituation-Dishabituation Tests. Bio-protocol 5(13): e1518. DOI: 10.21769/BioProtoc.1518.
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