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Locomotion Activity Measurement in an Open Field for Mice

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Frontiers in Behavioral Neuroscience
Jul 2015



The Open Field (OF) paradigm is one of the most known primary behavioral tests to evaluate locomotion and exploration. Altered OF behaviour is relatively simple to observe, but understanding and explaining the reasons for the observed changes is a complex task. Generally, there are two factors, which determine the behavior in this paradigm; one, a positive exploratory drive originating from the nature of rodents to explore new environments (for food and shelter); and two, the animal nature of avoiding open and brightly lit spaces (exposure to predators).

OF measures locomotor activity, exploratory drive, neophobia and certain aspects of anxiety in rodents at the same time. Furthermore, one can differ between horizontal and vertical activities (number of rearings) in the OF. After all, an altered OF behavior might come from the alterations of all of the above mentioned measures. For the proper interpretation of experimental results one has to be careful.

With the aid of the present protocol we investigated the effect of systemic L-kynurenine sulphate on open field behavior of adult male C57Bl/6j mice (Varga et al., 2015).

Keywords: Open field behavior (旷场行为), Moving pattern (运动模式), MANOVA (多元方差分析), Animal handling (动物处理), Digital video tracking (数字视频跟踪)

Materials and Reagents

  1. Adjustable dim light illumination is supplied with conventional argon light tubes
  2. Custom-made OF box prepared from common household plexiglass (Figure 1)
  3. Latex gloves
  4. 8-10-week-old male C57Bl/6j mice (n = 55) weighing 20-26 g
    Note: The animals were housed under controlled laboratory conditions, in groups of 5, under an inverse 12-h dark/light cycle, with ad libitum access to food and tap water. To avoid the effects of shipping stress a 2-week habituation period was used before initiation of the behavioral testing. All housing and experiments were conducted in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII.tv. 32. §). Efforts were made to minimize the number of animals used and to reduce pain and discomfort.

    Figure 1. Custom-made OF box prepared from common household plexiglass. Digital video tracking can be performed with a conventional coloured CCD camera fixed and positioned above the OF arena with a telescopic tripod stand. 


  1. Video-tracking system (Harvard Apparatus, model: SMART® v 3.0 ) connected to personal computer.
  2. Coloured CCD camera (Sony, model: SSC-DC378P )
  3. Telescopic tripod stand (Manfrotto, model: 420b )


  1. Multivariate ANOVA (mANOVA) (IBM Corporation, SPSS Statistics version 20)
  2. SMART® software


  1. General rules for OF set-up
    1. Illumination is one of the most important extrinsic factors in this test. Always measure illumination conditions (generally measured in Lux) in every point of the OF, avoid unintentional uneven illumination. 

    2. Avoid light reflections. It influences the behavior and possibly the tracking procedure too. 

    3. Cover the internal surface of the OF walls with matte colours. Light grey enables the easy detection of various coat colours if a video-tracking system is used. 

    4. Avoid external and internal visual cues. It may result in uneven place preference. 

    5. To avoid internal olfactory cues wipe the arena (usually 50% EtOH) before the running of the first subject and then between subjects carefully. Urination and defecation cues may change the exploratory pattern of the next animal. Before the next trial let 50% EtOH completely dry up.
    6. Bottom of the OF arena should be mildly rough to enable animals to move without sliding.
    7. Arena shape and size can be designated individually (Eilam, 2003; Lebedev et al., 2012).
    8. For general mouse handling conditions see Hurst and West, 2010.
    9. Mind the diurnal cycle of the mice. Mice are nocturnal animals. One can possibly work either in the active or in the inactive phase of the animals, but experiments must be timed in the same narrow time window of the day.
    10. Make video recording, perform off-line data analysis.
    11. Make a distinction between center and peripheral parts of the OF either manually on the bottom of the OF or in a computer-aid manner.
    12. Measure weight and check general health conditions of the animals one day before the OF experiment.
      Note: Below, we describe a complete protocol, which we used for assessing the behavioral changes in OF after systemic L-kynurenine sulphate treatment (Varga et al., 2015).

  2. Locomotion activity measurement in an open field
    1. The OF consisted of a square arena (50 x 50 cm) enclosed by continuous, 50-cm-high, light-gray opaque walls made of plexiglass.
    2. The apparatus is placed in a room illuminated by adjustable lamps giving a dim light within the arena (around 280 lux).
    3. Mice (n = 9 per control group, 10 per L-KYNs-treated group) are placed into the middle of one side of the arena facing the wall.
    4. The animals are allowed to move freely for 8 min, while their horizontal ambulatory activity is tracked with the aid of a video-tracking system (see tracked moving path of a control animal in Figure 2). This allows us to measure all the required parameters: total distance moved (cm), time spent moving (s), average speed (cm/s) and proportion of total time spent in the OF arena in different speed threshold ranges (percentage). Speed thresholds are correlated to the maximal moving speed (45 cm/s) of the mice, previously determined with an independent cohort of animals. The maximal moving speed of the mice is then halved, to give a slow moving speed group (< 22.5 cm/s) and a high moving speed group (> 22.5 cm/s).
    5. Following the experimental session, the mice are carefully removed from the OF, and returned to their home cage. The test equipment is cleaned with 50% ethanol solution and dried between subjects in order to avoid olfactory cuing.

      Figure 2. Representative moving trace of a control mouse in the Open Field box. Peripheral zone preference (i.e., preference for being in the proximity of the walls) emerges. Moving pattern was registered with Video-tracking system.

  3. Behavioral observations in an open field
    1. The anxiety-like behavior in the OF arena is assessed. The percentage of the time spent within the central part of the arena is determined, which is illuminated slightly better (300 lux) than the peripheral parts (250 lux). The central area delineated virtually with SMART® software, is taken as an imaginary inner square (30 x 30 cm) of the OF. The 8-min free exploration period is recorded simultaneously by a video recorder. Stereotyped behavior relevant at the level of anxiety (number of rearings, and times spent grooming and freezing) are scored manually (Carola et al., 2002). A single primary observer blind to the experimental condition conducts the behavioral observations.
    2. Statistical analysis of the OF behavior is performed with the multivariate ANOVA (mANOVA) (If the data distribution violates the assumptions of parametric tests, a non-parametric test (e.g., Kruskal-Wallis test) must be used.).
    3. For video record please visit: http://journal.frontiersin.org/file/downloadfile/292440/octet-stream/Video%201.WMV/7/3/144405.


Digital video tracking is performed with a conventional coloured CCD camera fixed and positioned above the OF arena with a telescopic tripod stand (Figure 1). PC is installed in an adjacent room, so mice are unperturbed during the whole experiment (This separation is recommended for all kinds of behavioral experiments.).


This study was supported by grant OTKA K105077 and cofinanced by the EUROHEADPAINFP7-Health 2013-Innovation; Grant No. 602633, and grant by MTA-SZTE Neuroscience Research group.


  1. Carola, V., D'Olimpio, F., Brunamonti, E., Mangia, F. and Renzi, P. (2002).  Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice.Behavioural Brain Research 134(1-2): 49-57.
  2. Eilam, D. (2003). Open-field behavior withstands drastic changes in arena size. Behav Brain Res 142(1-2): 53-62.
  3. Hurst, J. L. and West, R. S. (2010). Taming anxiety in laboratory mice. Nat Methods 7(10): 825-826.
  4. Lebedev, I. V., Pleskacheva, M. G. and Anokhin, K. V. (2012). [C57BL/6 mice open field behaviour qualitatively depends on arena size]. Zh Vyssh Nerv Deiat Im I P Pavlova 62(4): 485-496.
  5. Varga, D., Heredi, J., Kanvasi, Z., Ruszka, M., Kis, Z., Ono, E., Iwamori, N., Iwamori, T., Takakuwa, H., Vecsei, L., Toldi, J. and Gellert, L. (2015). Systemic L-Kynurenine sulfate administration disrupts object recognition memory, alters open field behavior and decreases c-Fos immunopositivity in C57BI/6 mice. Frontiers in Behavioral Neuroscience 9: 14-24.



关键字:旷场行为, 运动模式, 多元方差分析, 动物处理, 数字视频跟踪


  1. 可调节的暗光照明与常规氩光管一起提供
  2. 定制的由普通家用有机玻璃制成的盒子(图1)
  3. 乳胶手套
  4. 体重为20-26g的8-10周龄雄性C57Bl/6j小鼠(n = 55) 注意:将动物在受控的实验室条件下,以5只为一组,在反向12小时黑暗/光照循环下饲养,随意获得食物和自来水。为了避免运送压力的影响,在开始行为测试之前使用2周的适应期。所有住房和实验都按照欧洲共同体委员会指令(86/609/ECC)和匈牙利保护动物研究法案(XXVIII.tv.32§)进行。努力使使用的动物数量最小化并减少疼痛和不适。



  1. 连接到个人计算机的视频跟踪系统(Harvard Apparatus,型号:SMART ? v 3.0)。
  2. 彩色CCD相机(Sony,型号:SSC-DC378P)
  3. 可伸缩三脚架(Manfrotto,型号:420b)


  1. 多变量ANOVA(mANOVA)(IBM公司, SPSS Statistics第20版
  2. SMART ?软件


  1. 设置的一般规则
    1. 照明是本测试中最重要的外在因素之一。总是在OF的每个点测量照明条件(通常以Lux测量),避免无意的不均匀照明。
    2. 避免光反射。它也影响行为和可能的跟踪过程。
    3. 用哑光色覆盖OF墙的内表面。浅灰色使得能够在使用视频跟踪系统的情况下容易地检测各种涂层颜色。
    4. 避免外部和内部视觉线索。它可能导致不均匀的地方偏好。
    5. 为了避免内部嗅觉提示,在运行第一个受试者之前,然后在受试者之间仔细地擦拭竞技场(通常50%EtOH)。排尿和排便线索可能改变下一个动物的探索模式。在下次试验之前,让50%EtOH完全干透。
    6. OF舞台的底部应该略微粗糙,以使动物能够不滑动地移动
    7. 竞技场的形状和大小可以单独指定(Eilam,2003; Lebedev等人,2012)。
    8. 对于一般鼠标处理条件,请参阅Hurst和West,2010年。
    9. 注意小鼠的昼夜周期。小鼠是夜间动物。人们可能在动物的活动期或非活动期工作,但实验必须在一天的相同的狭窄时间窗内计时。
    10. 进行视频录制,进行离线数据分析。
    11. 在OF的底部手动地或以计算机辅助的方式区分OF的中心部分和外围部分。
    12. 在OF实验前一天测量体重并检查动物的一般健康状况。

  2. 在开放领域的运动活动测量
    1. OF包括由连续的,50cm高,由有机玻璃制成的浅灰色不透明壁包围的正方形竞技场(50×50cm)。
    2. 将装置放置在由可调灯照亮的房间中,在场所内给出暗光(约280lux)。
    3. 小鼠(每个对照组n = 9,每个L-KYNs治疗组10只)被放置在面向壁的体育场的一侧的中间。
    4. 允许动物自由移动8分钟,同时借助于视频跟踪系统(参见图2中对照动物的跟踪的移动路径)来跟踪它们的水平走动活动。这允许我们测量所有需要的参数:移动的总距离(cm),移动时间,平均速度(cm/s)和在不同速度阈值范围(百分比)中在OF领域中花费的总时间的比例。速度阈值与小鼠的最大移动速度(45cm/s)相关,先前用独立的动物组确定。然后将小鼠的最大移动速度减半,得到缓慢移动速度组(<22.5cm/s)和高移动速度组(> 22.5cm/s)。
    5. 在实验期后,小心地从OF中取出小鼠,并返回到它们的家笼。测试设备用50%乙醇溶液清洁并在受试者之间干燥以避免嗅觉。


  3. 行为的 在开放字段中的观察
    1. 评估OF场中的焦虑样行为。确定在竞技场的中心部分内花费的时间的百分比,其比周边部分(250勒克斯)稍好地照亮(300lux)。使用SMART ?软件虚拟描绘的中心区域被视为OF的假想内部正方形(30×30cm)。 8分钟的自由探测期由录像机同时记录。手工评分焦虑水平相关的定型行为(回访次数,和修饰和冷冻所花费的时间)(Carola等人,2002)。对实验条件失明的单个主观察者进行行为观察。
    2. 用多变量ANOVA(mANOVA)进行OF行为的统计分析(如果数据分布违反参数检验的假设,则必须使用非参数检验(例如, Kruskal-Wallis检验) 。)。
    3. 对于视频记录,请访问: http://journal.frontiersin.org/file/downloadfile/292440/octet-stream/Video%201.WMV/7/3/144405


数字视频跟踪是用常规的彩色CCD照相机进行的,该照相机用可伸缩的三脚架(图1)固定并定位在OF场的上方。 PC安装在相邻的房间,所以小鼠在整个实验期间不受干扰(这种分离被推荐用于各种行为实验)。


这项研究得到了OTKA K105077的资助,由EUROHEADPAINFP7-Health 2013-Innovation共同资助;拨款号602633,由MTA-SZTE神经科学研究组授予。


  1. Carola,V.,D'Olimpio,F.,Brunamonti,E.,Mangia,F.and Renzi,P。(2002) 高架加迷宫和开放场测试的评估评估近交小鼠中焦虑相关行为。 行为 134
  2. Eilam,D。(2003)。  开放场行为可抵抗竞技场尺寸的剧烈变化。 Behav Brain Res 142(1-2):53-62。
  3. Hurst,JL and West,RS(2010)。  Taming实验室小鼠的焦虑。国家方法 7(10):825-826。
  4. Lebedev,IV,Pleskacheva,MG and Anokhin,KV(2012)。  [C57BL/6小鼠开放场行为定性地取决于竞技场大小]。 Zh Vyssh Nerv Deiat Im IP Pavlova 62(4):485-496。
  5. Varga,D.,Heredi,J.,Kanvasi,Z.,Ruszka,M.,Kis,Z.,Ono,E.,Iwamori,N.,Iwamori,T.,Takakuwa,H.,Vecsei, Toldi,J.和Gellert,L.(2015)。  系统性L-犬尿氨酸硫酸盐给药破坏了对象识别记忆,改变了开放场行为并降低了C57BI/6小鼠的c-Fos免疫阳性。行为神经科学的前沿9:14-24。
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引用:Gellért, L. and Varga, D. (2016). Locomotion Activity Measurement in an Open Field for Mice. Bio-protocol 6(13): e1857. DOI: 10.21769/BioProtoc.1857.