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Olfactory Recognition Memory Test in Mice
小鼠嗅觉认知记忆测试   

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Abstract

Olfactory memory is an ethologically relevant task that relies on a mouse’s innate ability to use olfaction to forage for food (Zou et al., 2015), and identify safe foods. Although many of the same brain areas involved in other forms of memory are also involved in olfactory memory, the mechanisms are different (Sanchez-Andrade et al., 2005; Tong et al., 2014). Here, we describe one way to test olfactory memory in mice. The protocol described can be used to test long-term memory (memory which requires de novo protein synthesis) or short term memory by adjusting the delay time between the training session and the recall session (Freedman et al., 2013) and has been designed to mimic the single presentation of the social recognition paradigm. This paradigm relies on the mouse’s innate tendency to investigate a novel scent more than a familiar scent. Transgenic NR2A overexpression mice are known to have impaired long-term olfactory memory, but intact short-term memory, and are used here to demonstrate how one form of impaired olfactory memory may appear. Other genetically or chemically manipulated mice may be used in place of the transgenic mice used here.

Materials and Reagents

  1. Filter paper (Bio-Rad Laboratories, catalog number: 1703967 )
  2. Pipet or syringe (BD, catalog number: 309625 )
  3. Low odor epoxy or glue, or tape to seal the scent cup
  4. Adult 3-8 months old mice, for the testing group (transgenic NR2A overexpression mice) and wild type mice for the control group (referred to as “subject mouse”)
  5. 70% ethanol solution
  6. Fresh or canned, non-expired 100% juice samples [pineapple (Dole), orange (Tropicana), 100% lemon and 100% lime juices] (see Figure 1)

Equipment

  1. Clean mouse cages identical to the home cage (7.5 in x 11 in x 5 in), one for each subject mouse per phase
  2. Clear Plexiglas to cover the cage top (so the mice cannot climb out)
  3. Small plastic container with a lid (i.e., a plastic petri dish, 35 mm x 10 mm) (Thermo Fisher Scientific, catalog number: FB0875711YZ , or other similar replacement)
  4. Drill with small (1 mm) drill bit or 18 g needle (BD, catalog number: 305196 ) for making holes in top of scent cup
  5. Digital recording camera
  6. Laboratory timer
  7. Stopwatch for recording investigation times


    Figure 1. Experimental set-up for the olfactory recognition task. For the olfactory recognition task, a selection of juices (pineapple, orange, lemon and lime), a small Petri dish, a syringe and an empty clean mouse cage are used.

Procedure

  1. Experimental setup
    1. Experimental space should be quiet and dimly lit (75 to 100 lux) to avoid distraction from sounds and increased anxiety level from bright lights.
    2. The experimental area is surrounded by a back curtain with the experimenter outside of the curtain to avoid distraction from movements.
    3. A digital recording camera is mounted overhead and connected to a computer for recording the experiment.
    4. The olfactory recognition experiment has two phases, a training phase and a recall phase. Long-term memory can be tested by delaying the recall phase for 24 h. Short-term memory can be tested by employing a short delay time between the two phases, such as one hour.
    5. Create the scent cup by drilling four or five small holes in the lid of the container. Place a piece of the filter paper inside the container and seal the container using low odor glue or epoxy, or tape.

  2. Olfactory recognition
    1. Training phase
      1. Place the subject mouse into a clean cage, identical to the home cage, with the scent cup for 30 min prior to testing to allow the animal to habituate to the cage and the scent cup.
      2. Briefly remove the scent cup, place 0.10 ml of fruit juice on the filter paper inside the scent cup with a syringe or pipet and place the scent cup back into the cage. For guidance on placement within the cage see Figure 2.
      3. Allow the mouse to freely explore the scent, after 5 min, place the mouse back into its home cage.
    2. Recall phase
      1. For the recall phase, split the subject mice into two groups. One group will be paired with a novel fruit juice (this is the control group), the novel juice should be explored to a similar extent as the juice in the training phase. This control group demonstrates that the decrease in exploration is not due to fatiguing effects. The second group will be paired with the same fruit juice as in the training phase (this is the experimental group).
      2. Place the subject mouse into a clean mouse cage, containing a scent cup for 30 min prior to testing.
      3. Briefly remove the scent cup, place 0.10 ml of the appropriate fruit juice into the scent cup and return the scent cup back into the cage with the subject mouse for 5 min. For guidance on placement within the cage see Figure 2.
      4. After 5 min, the subject mouse is placed back into the home cage.

  3. Data analysis
    1. Using a stopwatch, have a blind experimenter record the amount of time that the mouse spends investigating the scent. Investigation of the scent is defined as the mouse being within one centimeter of the scent cup with its head directed toward the scent cup (Figure 2, Video 1). Alternatively, there are several video tracking software systems that can be used (i.e., BIOBSERVE software), however if bedding is used in the testing cage, as in this protocol, the scent cup may move. If automated tracking software is used, it would be best to secure the scent cup to the cage floor.


      Figure 2. Data analysis for the olfactory recognition task. A. Diagram illustrating the investigation criteria of the scent. B. Picture of a subject mouse investigating a scent.

      Video 1. Video clip of the olfactory recognition task

    2. Record the time that the animal spends investigating the scent cup in seconds.
    3. Group the animals according to the genotype, sex, or treatment group.
    4. Average the exploration times and determine the standard error.
    5. Plot the data using the standard error measurements as the error bars.
    6. Differences between the training session and the recall session can be determined using a Student’s t-test. The animal is said to have formed a memory of the scent if there is a significant decrease in the amount of time spent investigating the familiar scent from the training session to the recall session. Differences among groups can be determined by an ANOVA.

Representative data

Figure 3 is representative data for a 24-h olfactory recognition memory task. In this task, the mice were introduced to a fruit scent in the first round and were allowed to investigate the fruit scent for 5 min. The mice were then split into two groups; one group will be presented with the identical scent, the other group will be presented with a novel scent. The wild type mice are able to learn and recall the familiar scent, as evident by the significant decrease (as determined by a Student’s t-test) in the exploration of the familiar scent in the recall phase. The NR2A mice (used here to demonstrate impaired olfactory memory) are unable to recall the familiar scent in the 24 h recall phase and did not significantly reduce the exploration times of the familiar scent.


Figure 3. Representative data for a 24 h long-term olfactory recognition task. A. Representative exploration times, in seconds, of the juice for both the wild type and the NR2A transgenic mice. The identical scent was used in both sessions. B. Graphical representation of the averages of the exploration times. C. Representative exploration times, in seconds, of the juices for both the wild type and the NR2A transgenic mice. A novel juice was presented in the recall session. D. Graphical representation of the averages of the exploration times with. A novel juice scent was presented in the recall session.

Notes

  1. Experimental setup should be in a slightly darkened, quiet room as mice often show fear of brightly lit area and can be startled by sudden loud noises or movements (animal may freeze or avoid venturing away from the walls in response to fearful events) (Liu et al., 2014). Similarly, the animals should be habituated to the testing area (i.e., Ten minutes a day for 3 days prior to testing if standard houses cages are not used) and the empty scent cup (as described) to avoid the animals displaying neophobia (Deacon, 2006).
  2. If the mice display fear during the task, as a result of not being habituated to the caging or the scent cup, the investigation times will be reduced skewing the results. However, using the described measures, the animal should be habituated and not display signs of fear. If enclosures identical to the home cages are not used, longer habituation times may be required.
  3. Scent cups can be made to be reusable (i.e., with a screw-top cap). If reusable cups are use, clean the scent cups thoroughly between scents, sessions and animals to avoid scent from other animals contaminating the scent cup and skewing the results. Disposable scent cups can also be sealed with a small bead of low-odor glue or epoxy, or tape. Allow sufficient drying time for the glue or epoxy to avoid a strong scent.
  4. After the training session, the delay used before the recall session determined the type of memory tested. To test short term memory, use a short delay, such as 1 h. Short-term memory is memory which does not require protein synthesis. Twenty-four hours can be used to test long-term memory as 24 h memory has been found to require de novo protein synthesis.
  5. The number of animals used will depend on the strength of the manipulation to the subject animals. For many behavioral paradigms it is best to use n ≥ 10 for each group to ensure accuracy.

Acknowledgments

This work was supported by funds from the National Institute of Mental Health (MH060236), National Institute on Aging (AG024022, AG034663 & AG025918), USAMRA00002, and Georgia Research Alliance (all to JZT). This protocol was adapted from (Jacobs and Tsien, 2014; Jacobs et al., 2015).

References

  1. Deacon, R. M. (2006). Housing, husbandry and handling of rodents for behavioral experiments. Nat Protoc 1(2): 936-946.
  2. Freedman, K. G., Radhakrishna, S., Escanilla, O. and Linster, C. (2013). Duration and specificity of olfactory nonassociative memory. Chem Senses 38(4): 369-375.
  3. Jacobs, S. A. and Tsien, J. Z. (2014). Overexpression of the NR2A subunit in the forebrain impairs long-term social recognition and non-social olfactory memory. Genes Brain Behav 13(4): 376-384.
  4. Jacobs, S., Wei, W., Wang, D. and Tsien, J. Z. (2015). Importance of the GluN2B carboxy-terminal domain for enhancement of social memories. Learn Mem 22(8): 401-410.
  5. Liu, J., Wei, W., Kuang, H., Tsien, J. Z. and Zhao, F. (2014). Heart rate and heart rate variability assessment identifies individual differences in fear response magnitudes to earthquake, free fall, and air puff in mice. PLoS One 9(3): e93270.
  6. Sanchez-Andrade, G., James, B. M. and Kendrick, K. M. (2005). Neural encoding of olfactory recognition memory. J Reprod Dev 51(5): 547-558.
  7. Tong, M. T., Peace, S. T. and Cleland, T. A. (2014). Properties and mechanisms of olfactory learning and memory. Front Behav Neurosci 8: 238.
  8. Zou, J., Wang, W., Pan, Y. W., Lu, S. and Xia, Z. (2015). Methods to measure olfactory behavior in mice. Curr Protoc Toxicol 63: 11 18 11-21.

简介

嗅觉记忆是一种与病因相关的任务,其依赖于小鼠使用嗅觉来觅食的固有能力(Zou等人,2015),并且识别安全食物。尽管参与其他形式记忆的许多相同的脑区域也参与嗅觉记忆,但是机制是不同的(Sanchez-Andrade等人,2005; Tong等人, em>,2014)。在这里,我们描述了一种方法来测试小鼠的嗅觉记忆。所描述的方案可以用于通过调节训练期和召回期之间的延迟时间来测试长期记忆(需要重新编码蛋白质合成的记忆)或短期记忆。(Freedman < et al。,2013),并且被设计为模仿社会识别范例的单一表示。这种模式依赖于小鼠的天生倾向来研究一种新颖的香味而不是一种熟悉的香味。转基因NR2A过表达小鼠已知受损的长期嗅觉记忆,但完整的短期记忆,并在这里用于演示如何一种形式的受损嗅觉记忆可能出现。可以使用其他遗传或化学操作的小鼠来代替本文使用的转基因小鼠。

材料和试剂

  1. 滤纸(Bio-Rad Laboratories,目录号:1703967)
  2. 移液管或注射器(BD,目录号:309625)
  3. 低气味环氧树脂或胶水,或胶带密封香杯
  4. 用于测试组(转基因NR2A过表达小鼠)的成年3-8个月大的小鼠和对照组的野生型小鼠(称为"受试小鼠")
  5. 70%乙醇溶液
  6. 新鲜或罐装,未过期的100%果汁样品[菠萝(Dole),橙(Tropicana),100%柠檬和100%柠檬汁](见图1)

设备

  1. 清洁与家笼相同的鼠笼(7.5英寸x 11英寸x 5英寸),每个阶段每个主题小鼠一个
  2. 清除有机玻璃覆盖笼顶(因此小鼠不能爬出来)
  3. 带有盖子(即塑料培养皿,35mm×10mm)(Thermo Fisher Scientific,目录号:FB0875711YZ,或其他类似替换物)的小塑料容器
  4. 用小(1 mm)钻头或18 g针(BD,目录号:305196)钻孔,在气味杯顶部打孔
  5. 数字录像机
  6. 实验室定时器
  7. 记录调查时间的秒表


    图1.嗅觉识别任务的实验设置。 对于嗅觉识别任务,使用选择的果汁(菠萝,橙,柠檬和酸橙),小培养皿,注射器和空的清洁鼠标笼。

程序

  1. 实验设置
    1. 实验空间应安静,昏暗(75至100勒克斯) 避免从声音分心,增加焦虑水平从明亮 灯。
    2. 实验区被一个后幕包围 与实验者外面的窗帘,以避免分心 运动。
    3. 数字记录摄像机安装在顶部并连接到计算机用于记录实验
    4. 嗅觉识别实验有两个阶段,一个训练 阶段和回忆阶段。 长期记忆可以通过延迟测试回忆期24 h。 短期记忆可以通过使用a 两个相之间的短延迟时间,例如一个小时。
    5. 通过在盖子上钻四个或五个小孔来创建气味杯 容器。 将一块滤纸放在容器中 并使用低气味的胶或环氧树脂或胶带密封容器。

  2. 嗅觉识别
    1. 训练阶段
      1. 将主题鼠标放入一个干净的笼子里,相同的家笼,与气味杯30分钟前 测试以允许动物习惯于笼子和香味杯。
      2. 短暂取出香味杯,在上面放入0.10ml果汁 用注射器或移液管在气味杯内部放置滤纸并放置 香味杯回到笼子里。 关于在笼内放置的指导 见图2.
      3. 让鼠标自由探索气味,5分钟后,将鼠标放回其家笼中。
    2. 调用阶段
      1. 对于召回阶段,将受试小鼠分成两组。 一组将与一个新的果汁配对(这是控制 组),新鲜果汁应该探索与类似的程度 果汁在训练阶段。 这个控制组表明 降低勘探不是由于疲劳效应。 第二 组将与在训练阶段与相同的果汁配对 (这是实验组)。
      2. 在测试前,将受试小鼠放入干净的鼠笼中,含有香杯30分钟。
      3. 短暂取出香味杯,放入0.10ml适当的水果   果汁进入香杯,并将香杯回到笼子里 与对象小鼠5分钟。 关于内部放置的指导 笼子见图2.
      4. 5分钟后,将受试小鼠放回家笼中。

  3. 数据分析
    1. 使用秒表,有盲实验者记录的时间量 小鼠花费调查气味。 调查 气味被定义为小鼠在气味的一厘米内 杯子,其头部指向香味杯(图2,视频1)。 或者,存在若干视频跟踪软件系统 可以使用(即 BIOBSERVE软件),但是如果使用床上用品   测试笼,如在本协议中,香味杯可能移动。 如果自动   使用跟踪软件,最好将香味杯固定 笼式地板。


      图2.嗅觉的数据分析 识别任务。 A.说明调查标准的图表 香味。 B.调查气味的主题老鼠的照片。

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    2. 记录动物花费几秒钟调查气味杯的时间。
    3. 根据基因型,性别或治疗组分组动物
    4. 平均勘探时间并确定标准误差。
    5. 使用标准误差测量值绘制数据作为误差条。
    6. 训练会话和召回会话之间的差异可以 使用Student's T检验确定。 据说动物有 形成了香味的记忆,如果有明显的减少 花费在调查熟悉的气味从训练花费的时间 会话到召回会话。 组之间的差异可以 通过ANOVA确定。

代表数据

图3是24小时嗅觉识别记忆任务的代表性数据。在该任务中,在第一轮中将小鼠引入水果香味,并允许研究水果香味5分钟。然后将小鼠分成两组;一组将呈现相同的香味,另一组将呈现一种新颖的香味。野生型小鼠能够学习和回忆熟悉的香味,通过在回忆阶段对熟悉的香味的探索中的显着减少(通过学生t检验确定)是明显的。 NR2A小鼠(用于证明受损的嗅觉记忆)在24小时召回阶段不能回忆熟悉的气味,并且没有显着减少熟悉的香味的探索时间。


图3.24小时长期嗅觉识别任务的代表性数据 A.野生型和NR2A转基因小鼠的果汁的代表性探索时间(秒)。在两个会话中使用相同的香味。 B.图形代表勘探时间的平均值。 C.野生型和NR2A转基因小鼠的果汁的代表性探索时间,以秒计。在召回会议中提出了一种新颖的果汁。 D.图形代表勘探时间的平均值。在召回会议中提出了一种新颖的果汁香味。

笔记

  1. 实验设置应该在一个稍微变暗,安静的房间,因为老鼠常常表现出对明亮的光线区域的恐惧,并可以突然大声的噪音或运动(动物可能会冻结或避免冒险远离墙壁响应恐怖事件) et al。,2014)。类似地,动物应该习惯于测试区域(即,如果不使用标准房舍笼子,则在测试之前每天十分钟,持续3天)和空的香味杯子(如所述)以避免动物表现出恐惧症(Deacon,2006)。
  2. 如果小鼠在任务期间显示恐惧,由于不习惯于笼中或气味杯,调查时间将减少,结果偏差。然而,使用所述措施,动物应该习惯,并且不显示恐惧的迹象。如果不使用与家笼相同的罩,则可能需要较长的适应时间。
  3. 可以使气泡杯可重复使用(,即带有螺旋盖)。如果使用可重复使用的杯子,在气味,会话和动物之间彻底清洁气味杯,以避免其他动物的香味污染气味杯和歪斜结果。一次性香味杯也可以用小的低气味胶或环氧树脂或胶带密封。为胶水或环氧树脂留出足够的干燥时间,以避免强烈的气味。
  4. 在训练会话之后,在召回会话之前使用的延迟确定测试的存储器的类型。要测试短期记忆,使用短暂的延迟,如1小时。短期记忆是不需要蛋白质合成的记忆。 24小时可用于测试长期记忆,因为已发现24小时记忆需要蛋白质合成。
  5. 使用的动物数量将取决于对受试动物的操作强度。 对于许多行为范式,最好对每个组使用n≥10以确保准确性

致谢

这项工作由国家心理健康研究所(MH060236),国家老龄化研究所(AG024022,AG034663& AG025918),USAMRA00002和佐治亚研究联盟(全部给JZT)的资金支持。 该协议改编自(Jacobs和Tsien,2014; Jacobs等人,2015)。

参考文献

  1. Deacon,R.M。(2006)。 针对行为实验的啮齿类动物的饲养,饲养和处理。<\ n>/em> 1(2):936-946。
  2. Freedman,K.G.,Radhakrishna,S.,Escanilla,O。和Linster,C。(2013)。 嗅觉非关联记忆的持续时间和特异性 化学感觉 38(4):369-375。
  3. Jacobs,S.A。和Tsien,J.Z.(2014)。 NR2A亚基在前脑中的过表达损害长期社会认知和非社会嗅觉记忆。 Brain Behav 13(4):376-384。
  4. Jacobs,S.,Wei,W.,Wang,D.and Tsien,J.Z.(2015)。 GluN2B羧基末端结构域的重要性,用于增强社交记忆。 Learn Mem 22(8):401-410。
  5. Liu,J.,Wei,W.,Kuang,H.,Tsien,J.Z.and Zhao,F.(2014)。 心率和心率变异性评估可识别恐惧对地震,自由下降的反应幅度的个体差异, PLoS One 9(3):e93270。
  6. Sanchez-Andrade,G.,James,B.M.and Kendrick,K.M。(2005)。 嗅觉识别记忆的神经编码 J Reprod Dev 51(5):547-558
  7. Tong,M. T.,Peace,S.T.和Cleland,T.A。(2014)。 嗅觉学习和记忆的属性和机制 Front Behav Neurosci < em> 8:238。
  8. Zou,J.,Wang,W.,Pan,Y. W.,Lu,S.and Xia,Z.(2015)。 测量小鼠嗅觉行为的方法 Curr Protoc Toxicol 63:11 18 11-21。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jacobs, S. A., Huang, F., Tsien, J. Z. and Wei, W. (2016). Olfactory Recognition Memory Test in Mice. Bio-protocol 6(9): e1803. DOI: 10.21769/BioProtoc.1803.
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