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Novel Object Recognition for Studying Memory in Mice
采用新物体识别研究小鼠的记忆   

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Abstract

Memory tests are important indexes of the brain functions for rodents behavior assay. Many memory tasks require external forces (e.g. electric shocks) or intrinsic forces (e.g. hunger and thirsty) to trigger the responses. Under those conditions, rodents are under stresses, such as pain, tired, malnutrition or dehydration, which potentially affect the natural neural responses. Novel object recognition is a non-force driving and spontaneous memory test. It is derived from curiosity but easy to be interfered with manipulation factors. In addition to stepwise procedure, the protocol described here emphasizes how to reduce the noise in the novel object recognition.

Keywords: Mouse behavior(老鼠行为), Memory(记忆), Object recognition(物体识别)

Materials and Reagents

  1. Mouse
    Notes:
    1. To reach statistic significance, at least 10 mice are required. 15 mice are ideal to minimize the individual variation.
    2. C57BL/6, male, 2 months to one year old (2-4 months old is optimal), 3-5 mice are kept in one cage after weaning.
  2. Cleaning solution
    Note: Clean the chamber and/or objects using 70% ethanol first followed by sterile water (to remove the odor of ethanol). Air dry for 3 min.

Equipment

  1. Apparatus
    Note: The dimensions of plexiglas box is 40 cm x 40 cm x 40 cm. The color of plexiglas is frosted white or transparent. The frosted white plexiglas boxes can diminish the effect from outside views, if the mice have not experienced the open field test.


    Figure 1. The apparatus for novel object recognition

  2. Objects
    Note: The size of objects is 4 cm x 4 cm x 4 cm approximately. The shape of objects is simple like the building blocks. The objects made by wood, metal or hard plastic are preferred, because those are resistant to biting demolition. Because mice are protanope (red color blindness), the colors of objects should be checked by analysis of Vischeck software (a free software available from the website: http://www.vischeck.com) to ensure that mice are able to distinguish them.
  3. Camera/computer
    Note: The raw data are stored in recording equipment for further analysis. The video tracking system such as “Smart system” (Panlab) can help you to easily get the exploration time of mice to the objects (the heads of mice crossover the object boundary lines indicated in Figure 1). The exploration time can also be counted manually. The results are further analyzed using the Prism (Graph Pad software).

Procedure

Notes:

  1. The novel object recognition test is a non-forced recognition memory test. Any stresses or clues for mice can increase the variability of the results. It is very important to minimize the stimulation from environment and the objects and to make mice acclimating.
  2. Before testing, one pingpong ball is put into the home cage for a week to decrease mice’s stress induced by novel object.
  3. If the mice have not been subjected to any test, transfer them in an empty cage for 5 min per day for at least 3 days to reduce their stress responses in the future task.
  4. The plexiglas boxes and objects are cleaned by cleaning solutions and air-dried for 3 min before usage.
  5. The entire exploration process of mice is recorded using camera and stored in computer.

    1. Day 1-2: Habituation
      1. Mice are first transported to preparing area, which is a place near for the operative space for 10 min.
      2. Then the mice are put into the apparatus for analysis for 10 min to acclimate the manipulations and environment for decreasing their stress.
      3. This procedure is carried out for two days.

    2. Day 3: Training day
      1. Similar to Day 1 and 2, mice are first transported to preparing area, which is a place near for the operative space for 10 min.
      2. Two identical objects are placed in the plexiglas boxes as indicated in Figure 1 for mice to explore for 10 min.

    3. Day 4: Testing day
      All the processes are the same as the Day 3, except that one of old objects is randomly replaced with a new different object (Video 1).

      Video 1. Novel object

    4. Analysis
      1. The exploration time is counted by the period that mice stay on the circle area indicated in Figure 1.
      2. If the total of exploration time is less than 20 sec, mouse is likely under the stress. The data should be excluded.
      3. If the total of exploration time is more than 5 min, the recorded film needs to be checked by person. For instance, mouse may stand on the object or grooms on the circle area. The result should also be excluded.
      4. The preference rates are measured by dividing the total exploration time by the time to explore object. The formula to calculate the object preference is: the preference % = time to explore the individual object / total exploration time to objects x 100%.
      5. An example of novel object recognition is shown in Figure 2. At Day 3, the exploration times for two identical objects are similar (around 50%). At Day 4, animals show higher preference to the novel object. The mean of preference to the novel object is higher than 50% (~60% is commonly found). The one-way ANOVA using Prism is used for statistic analysis.


        Figure 2. The object preference at Day 3 and Day 4 of wild-type mice (*P < 0.05)

Acknowledgments

This protocol was adapted from the previous works Moscardo et al. (2012) and Huang et al. (2014).

References

  1. Huang, T. N., Chuang, H. C., Chou, W. H., Chen, C. Y., Wang, H. F., Chou, S. J. and Hsueh, Y. P. (2014). Tbr1 haploinsufficiency impairs amygdalar axonal projections and results in cognitive abnormality. Nat Neurosci 17(2): 240-247. 
  2. Moscardo, E., Salvetti, B., Becchi, S., Bertini, G. and Fabene, P. (2012). The novel object recognition test in rodents: which are the essential methodological aspects? Measuring Behavior: 476.

简介

记忆测试是啮齿动物行为测定的大脑功能的重要指标。 许多记忆任务需要外力(例如电击)或内在力(例如饥饿和渴望)来触发响应。 在这些条件下,啮齿动物处于压力下,例如疼痛,疲劳,营养不良或脱水,这可能影响自然神经反应。 新物体识别是一种非力驱动和自发记忆测试。 它源于好奇心,但容易受到操纵因素的干扰。 除了逐步过程,这里描述的协议强调如何减少新物体识别中的噪声。

关键字:老鼠行为, 记忆, 物体识别

材料和试剂

  1. 鼠标
    注意:
    1. 为了达到统计学意义,需要至少10只小鼠。 15只小鼠是理想的使个体变异最小化。
    2. C57BL/6,雄性,2个月至一岁(2-4个月龄是最佳的),3-5只小鼠在断奶后保持在一个笼子中。
  2. 清洁溶液
    注意:使用70%乙醇,然后用无菌水(以去除乙醇的气味)清洁室和/或对象。 空气干燥3分钟。

设备

  1. 装置
    注意:有机玻璃盒的尺寸为40厘米x 40厘米x 40厘米。有机玻璃的颜色是白色或透明的。如果老鼠没有经历旷场试验,磨砂的白色有机玻璃盒子可以减少外部视图的影响。


    图1.用于新对象识别的设备

  2. 对象
    注意:物体的大小约为4厘米x 4厘米x 4厘米。对象的形状很像建筑块。由木头,金属或硬塑料制成的物体是优选的,因为这些物体耐受咬住拆除。因为小鼠是protanope(红色盲),对象的颜色应该通过分析Vischeck软件(一个免费的软件可从网站:http://www.vischeck.com)检查,以确保小鼠能够区分 他们。
  3. 相机/电脑
    注意:原始数据存储在记录设备中以供进一步分析。 诸如"智能系统"(Panlab)的视频跟踪系统可以帮助您容易地获得小鼠到对象(小鼠的头越过图1中所示的对象边界线)的探索时间。 勘探时间也可以手动计算。 使用Prism(Graph Pad软件)进一步分析结果。

程序

注意:

  1. 新颖的对象识别测试是非强制识别记忆测试。 小鼠的任何压力或线索可以增加结果的变异性。 最小化来自环境和物体的刺激并使小鼠适应环境是非常重要的。
  2. 在测试之前,将一个乒乓球放入家笼中一周以减少小鼠由新物体诱导的应激。
  3. 如果小鼠没有经受任何测试,将它们在空笼子中每天转移5分钟至少3天以减少在未来任务中的应激反应。
  4. 有机玻璃盒和物体在使用前通过清洁溶液清洁并空气干燥3分钟。
  5. 鼠标的整个探索过程使用相机记录并存储在计算机中。

    1. 第1-2天:习惯
      1. 小鼠首先被运送到预备区域,这是靠近手术空间10分钟的地方。
      2. 然后将小鼠放入装置中进行分析10分钟                              适应环境以减少压力。
      3. 该程序进行两天。

    2. 第3天:训练日
      1. 类似于第1天和第2天,首先将小鼠运送到准备区域,其是靠近手术空间10分钟的地方。
      2. 两个相同的对象被放置在有机玻璃盒中,如图1所示,小鼠探索10分钟。

    3. 第4天:测试日
      所有过程与第3天相同,只是一个旧对象随机替换为一个新的不同对象(视频1)。

      视频1.新对象
                                                                                                                                                                                                                                                                                                       <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                                                                                                           

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >
                                                                                                                                                                                                                                                                                                       <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                                                                          

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >
    4. 分析
      1. 探索时间由小鼠停留在图1所示的圆圈区域的时间计数。
      2. 如果探测时间的总和小于20秒,小鼠可能处于压力下。 应排除数据。
      3. 如果探索时间总计超过5分钟,则记录胶片                             需要由人检查。例如,鼠标可能站在                             对象或新郎在圆形区域。结果也应排除。
      4. 优先率是通过总数除以                             探索时间由探索对象的时间。计算公式                             对象首选项是:首选项%=探索的时间                             单个对象/对对象的总探索时间x 100%。
      5. 在图2中示出了新的对象识别的示例。在第3天,                             两个相同对象的探索时间是相似的(周围                             50%)。在第4天,动物对新物体显示更高的偏好。的                             对新颖物体的偏好的平均值高于50%(〜60%                             常见)。使用Prism的单因素方差分析用于统计                             分析。


        图2.野生型小鼠的第3天和第4天的对象偏好(* P <0.05)

致谢

该协议改编自先前的作品Moscardo等人(2012)和Huang等人(2014)。

参考文献

  1. Huang,T.N.,Chuang,H.C.,Chou,W.H.,Chen,C.Y.,Wang,H.F.,Chou,S.J.and Hsueh,Y.P.(2014)。 Tbr1 haploinsufficiency impairs amygdalar 轴突投射和结果的认知异常。 Nat Neurosci 17(2):240-247。
  2. Moscardo,E.,Salvetti,B.,Becchi,S.,Bertini,G。和Fabene,P。(2012)。 啮齿类动物中的新型物体识别测试: 基本方法方面。测量行为:476.

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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, T. and Hsueh, Y. (2014). Novel Object Recognition for Studying Memory in Mice. Bio-protocol 4(19): e1249. DOI: 10.21769/BioProtoc.1249.
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