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Elevated Plus Maze Test to Assess Anxiety-like Behavior in the Mouse
高架十字迷宫试验评估小鼠的焦虑样行为   

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Abstract

The elevated plus maze task is a simple method to assess anxiety-like behaviors in rodents. This version describes the procedure used in mice. However, the protocol may also be applied to rats, considering a proportionally larger apparatus (arms: 10 x 50 cm; height: 55 cm). Briefly, the test is performed on a plus-shaped apparatus with two open and two closed arms. The animal is allowed to freely explore the maze for 5 min while the duration and frequency of entries into open and closed arms is recorded. The task is based on an approach-avoidance conflict, meaning that the animal is faced with a struggle between a propensity to explore a novel environment and an unconditioned fear of high and open spaces. Consequently, an anxiety-like state is characterized by increased open arm avoidance, compared to control animals. On account of being a very popular test, there can be considerable variations in the procedures applied across different laboratories. Here we provide a working protocol that has been able to detect both anxiogenic and anxiolyitic drug effects under the specified conditions. Protocol originally published in (Leo et al., 2014).

Materials and Reagents

  1. Laboratory-bred plus maze-naïve mice
    Note: Mice housed in groups of 4-5 per cage, kept in an environment with controlled temperature (around 23 °C) and humidity under a 12-12 h light-dark cycle with food and water ad libitum.
  2. Paper towels and 70% ethanol for cleaning

Equipment

  1. Elevated Plus maze (either customized apparatus or commercially available one)
  2. Video camera (placed directly above the maze)
  3. Indirect white light
  4. Digital lux meter (Instrutherm, Mastech, Dr. Meter or similar)
  5. Digital chronometers for manual analysis, or computer software (Any-maze, Ethovision or others) for automated analysis
    Note: In our laboratory, we use a custom made plus-shaped maze sized for mice (Leo et al., 2014), which is elevated 50 cm above the ground, and consists of two opposite closed arms, two opposite open arms and a central square of 5 cm sides (see image below). The closed arms measure 30 x 5 cm and are constituted of white-painted wood, as are the 15 cm high walls that enclose the arms. Glossy painting should be avoided as to prevent excessive glare, thus a matte finish is recommended. The open arms measure 30 x 5 cm and are made of transparent acrylic; a 0.3 cm high transparent acrylic railing prevents animals from falling while exploring the open arms. The apparatus can be disassembled and moved to and from the experiment room where it is set up for use. Wood-made apparatuses covered with wood lacquer, painted in black or transparent Plexiglas apparatuses are also frequently found. Commercially available apparatuses, such as those from Stoelting Co. or Panlab S.L.U., are recommended as alternatives. The room illumination and apparatus light reflection strongly affect the elevated plus maze test. In our laboratory, a Samsung Flashcam digital camcorder is used, as it is more appropriate for posterior off-line analysis. One should always run pilot experiments to establish reliable baseline parameters and standardize the experimental environment before starting the experimental manipulations (drugs, procedure, transgenic animals, etc.).


    Figure 1. Plus-shaped maze

Software

  1. Automated computer software (Any-maze, Ethovision and others)

Procedure

  1. The maze should be assembled in an isolated room away from any extraneous interference of noises, scents or movement. You may choose to place a source of low-intensity white noise in the behavioral experiment room. It should be noted that guidance cues, such as drawings on the walls around the room might skew the animal’s activity to a certain area of the maze. Therefore, any sort of paintings or artifacts that may serve as cues must be removed.
  2. The experimenter must restraint from making any excessive noise or movement during the entire trial and from wearing perfumes, colognes or any product with a strong smell, since it could act as anxiogenic stimulus for mice.
  3. Illumination in the room must be measured with the aid of a lux meter, kept constant and controlled according to the analysis that is to be performed. Given that low-intensity luminosity reduces open arm avoidance (Morato and Castrechini, 1989; Bertoglio and Carobrez, 2002; Cosquer et al., 2005; Pereira et al., 2005), to analyze an anxiogenic effect low-intensity lighting (5-30 Lux) should be preferred, whereas an anxiolytic effect should be analyzed under higher intensity lighting (200-400 Lux or more).
  4. After these experimental conditions are adjusted to a standard, the animals will be brought into the experiment room, where they will be left in their home cages for 45 to 60 min in order to recover from the stress of being moved.
  5. Clean the maze with 70% ethanol before starting the test in order to remove any dirt or smells accumulated on the apparatus.
  6. Turn on the video camera and place the first mouse in the center square of the maze facing one of the open arms, preferably the one opposite to the experimenter.
  7. The experimenter will stand as far away as possible from the maze and out of sight of the test animal, outside of the room if necessary. He must also avoid making unnecessary movement or sounds.
  8. After 5 min of free exploration, the mouse may be moved out of the maze and back into its home cage.
  9. All the urine and fecal boli must be removed and the maze cleaned entirely with 70% ethanol to remove any residual smell from the first mouse. Afterwards, the next mouse may be submitted to the test.
  10. Repeat steps 6-9 until all the animals have been tested.
  11. The recorded videos can be analyzed by automated computer software or manually with the aid of a chronometer. Several parameters can be considered:
    1. Entries in closed arms and time spent in closed arms: Measures of total locomotion on the maze throughout the experiment (Carola et al., 2002; Walf and Frye, 2007).
    2. Entries in open arms and time spent in open arms: May be used as inverse measures of anxiety, that is to say, reduced open arm avoidance reflects lower levels of anxiety (Carola et al., 2002; Walf and Frye, 2007; Griebel et al., 1993).
    3. Risk-assessment behavior: the frequency and the duration of head-dipping (downward movement of the head towards the floor while on the open arms) and stretch-attend posture (stretched posture with the head and two or three paws on the open arm and retraction to previous position) can be a direct measure of anxiety, i.e. increased risk-assessment behavior indicates higher levels of anxiety (Carola et al., 2002; Weiss et al., 1998; Carobrez and Bertoglio, 2005).
  12. Statistical analysis: Parameters should be examined separately by analysis of variance across all treatment schedules. The data can be further analyzed by a post-hoc test, if applicable.

Representative data



The figure above represents typical data from the elevated plus maze (EPM) in our settings, adapted to investigate anxiogenic responses (10 Lux). The behavior of the control group is relatively distributed, but decent statistically valid data has been gathered with a sample of 6-8 mice per group. Drug 1 is a clear anxiogenic drug, displaying decreased open arm time and open arm entries, without alterations in the time spent and number of entries in the closed arms. Drug 2 suggests a relatively flawed interpretation of an anxiogenic-like pharmacological profile, as treated-mice displayed decreased locomotion, as revealed by the number of entries in each arm. In such case, we recommend a slight reduction in drug dose in order to identify a dose allowing distinction between emotional and locomotor effects induced by the drug. A representative video from one of our experiments published in (Leo et al., 2014) is also available.

Notes

  1. Typically, male mice are used when performing behavioral experiments, as variations of estrous cycle often influence performance of females in such tests (Simpson et al., 2012; da Silva et al., 2014).
  2. Baseline activity may vary according to mouse strain, gender and age (Leo et al., 2014; Carobrez and Bertoglio, 2005; Hogg, 1996).
  3. Animals should be allowed at least one week for habituation to the home cage, as they tend to present high anxiety levels upon arrival from the animal breeder facility.
  4. In our experiments, animals are only handled for cage cleaning, weighing, drug treatment and during the testing. Also, animals are only tested during the light cycle; testing in the dark cycle may produce different results (Bertoglio and Carobrez, 2002).
  5. In order to ensure uniform luminosity, an indirect light should always be used to avoid the production of hard shadows, which can be a place of preference for the mouse, skewing its locomotion activity to that area of the maze. This may be done with an appropriate light reflector.
  6. The maze should always be cleaned after the testing of each animal.
  7. Make sure the maze is fully dried after cleaning, or a strong smell of the cleaning agent may alter mouse behavior.
  8. In the event that the mouse falls off the open arm during the test, the experimenter must immediately pick it up and place it back on the open arm. This should be noted and taken into consideration when performing the experiment analysis. This animal can be excluded from the final analysis.
  9. An entry in the open or closed arm is only considered when the animal places all four paws out of the center square and onto one of the arms. However, two paws out of a given arm, is already sufficient to stop counting the time spent into the arm. If the animal return with these two paws into the arm, the chronometer should be continued, but no entry is registered. A new entry is only registered after a full exit occurs.
  10. There is no evidence of age-related restrictions for behavioral testing in the elevated plus maze. Despite data supporting increased anxiety-like state in adolescent and aged animals (Lynn and Brown, 2010; Bessa et al., 2005; Chen et al., 2007), the task has been validated for successful assessment of anxiety-like behavior in adolescent, adult, middle-aged and aged rodents (Leo et al., 2014; Warneke et al., 2014; Carrasco et al., 2013; Joshi and Pratico, 2011; Pietrelli et al., 2012; Doremus et al., 2006).

Acknowledgments

The protocol described here has been adapted from a previous study (Leo et al., 2014), which succeeded in identifying anxiogenic-like as well as anxiolytic-like behavior for mice. The project received financial support from FAPERJ-Brasil (E-26/112.448/2012) and CNPq-Brasil (474443/2012-4).

References

  1. Bertoglio, L. J. and Carobrez, A. P. (2002). Behavioral profile of rats submitted to session 1-session 2 in the elevated plus-maze during diurnal/nocturnal phases and under different illumination conditions. Behav Brain Res 132(2): 135-143.
  2.  Bessa, J. M., Oliveira, M., Cerqueira, J. J., Almeida, O. F. and Sousa, N. (2005). Age-related qualitative shift in emotional behaviour: paradoxical findings after re-exposure of rats in the elevated-plus maze. Behav Brain Res 162(1): 135-142.
  3. Carobrez, A. P. and Bertoglio, L. J. (2005). Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 29(8): 1193-1205.
  4. 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. Behav Brain Res 134(1-2): 49-57.
  5. Carrasco, M. C., Vidal, J. and Redolat, R. (2013). Bupropion induced changes in exploratory and anxiety-like behaviour in NMRI male mice depends on the age. Behav Processes 98: 117-124.
  6. Chen, G. H., Wang, C., Yangcheng, H. Y., Liu, R. Y. and Zhou, J. N. (2007). Age-related changes in anxiety are task-specific in the senescence-accelerated prone mouse 8. Physiol Behav 91(5): 6.
  7. Cosquer, B., Galani, R., Kuster, N. and Cassel, J.-C. (2005). Whole-body exposure to 2.45 GHz electromagnetic fields does not alter anxiety responses in rats: a plus-maze study including test validation. Behav Brain Res 156(1): 65-74.
  8. da Silva, C. C., Lazzaretti, C., Fontanive, T., Dartora, D. R., Bauereis, B. and Gamaro, G. D. (2014). Estrogen-dependent effects on behavior, lipid-profile, and glycemic index of ovariectomized rats subjected to chronic restraint stress. Behav Processes 103: 327-333.
  9. Doremus, T. L., Varlinskaya, E. I. and Spear, L. P. (2006). Factor analysis of elevated plus-maze behavior in adolescent and adult rats. Pharmacol Biochem Behav 83(4): 570-577.
  10. Griebel, G., Moreau, J.-L., Jenck, F., Martin, J. R. and Misslin, R. (1993). Some critical determinants of the behaviour of rats in the elevated plus-maze. Behav Processes 29(1): 37-47.
  11. Hogg, S. (1996). A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav 54(1): 21-30.
  12. Joshi, Y. B. and Pratico, D. (2011). Knockout of 5-lipoxygenase results in age-dependent anxiety-like behavior in female mice. PLoS One 6(12): e29448.
  13. Leo, L. M., Almeida-Correa, S., Canetti, C. A., Amaral, O. B., Bozza, F. A. and Pamplona, F. A. (2014). Age-dependent relevance of endogenous 5-lipoxygenase derivatives in anxiety-like behavior in mice. PLoS One 9(1): e85009.
  14. Lynn, D. A. and Brown, G. R. (2010). The ontogeny of anxiety-like behavior in rats from adolescence to adulthood. Dev Psychobiol 52(8): 731-739.
  15. Morato, S. and Castrechini, P. (1989). Effects of floor surface and environmental illumination on exploratory activity in the elevated plus-maze. Braz J Med Biol Res 22(6): 707-710.
  16. Pereira, L. O., da Cunha, I. C., Neto, J. M., Paschoalini, M. A. and Faria, M. S. (2005). The gradient of luminosity between open/enclosed arms, and not the absolute level of Lux, predicts the behaviour of rats in the plus maze. Behav Brain Res 159(1): 55-61.
  17. Pietrelli, A., Lopez-Costa, J., Goni, R., Brusco, A. and Basso, N. (2012). Aerobic exercise prevents age-dependent cognitive decline and reduces anxiety-related behaviors in middle-aged and old rats. Neuroscience 202: 252-266.
  18.  Simpson, J., Ryan, C., Curley, A., Mulcaire, J. and Kelly, J. P. (2012). Sex differences in baseline and drug-induced behavioural responses in classical behavioural tests. Prog Neuropsychopharmacol Biol Psychiatry 37(2): 227-236.
  19. Walf, A. A. and Frye, C. A. (2007). The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protocols 2(2): 322-328.
  20. Weiss, S. M., Wadsworth, G., Fletcher, A. and Dourish, C. T. (1998). Utility of ethological analysis to overcome locomotor confounds in elevated maze models of anxiety. Neurosci Biobehav Rev 23(2): 265-271.
  21. Warneke, W., Klaus, S., Fink, H., Langley-Evans, S. C. and Voigt, J. P. (2014). The impact of cafeteria diet feeding on physiology and anxiety-related behaviour in male and female Sprague-Dawley rats of different ages. Pharmacol Biochem Behav 116: 45-54.

简介

高架+迷宫任务是评估啮齿动物焦虑样行为的一种简单方法。此版本描述了在小鼠中使用的程序。然而,考虑到成比例的更大的装置(手臂:10×50cm;高度:55cm),该方案也可以应用于大鼠。简言之,在具有两个开放臂和两个闭合臂的正形设备上进行测试。允许动物自由地探索迷宫5分钟,同时记录进入开放和闭合臂的持续时间和频率。任务是基于一种方法 - 回避冲突,意味着动物面临着探索一种新的环境的倾向和对高空和开放空间的无条件恐惧之间的斗争。因此,与对照动物相比,焦虑样状态的特征在于增加的开放臂避免。由于是一个非常受欢迎的测试,在不同实验室应用的程序可能有相当大的变化。在这里,我们提供一个工作协议,已能够检测焦虑和焦虑药物效果在指定的条件下。协议最初发表于(Leo em et al。,2014)。

材料和试剂

  1. 实验室繁殖加迷宫初级小鼠
    注意:将小鼠以每笼4-5只,保持在具有控制温度(约23℃)和湿度的环境中,在12-12小时光 - 暗循环下,食物和水随意进食。 em>
  2. 纸巾和70%乙醇用于清洁

设备

  1. Elevated Plus迷宫(定制设备或商用设备)
  2. 摄像机(直接放在迷宫上方)
  3. 间接白光
  4. 数字照度计(Instrutherm,Mastech,Dr. Meter或类似物)
  5. 用于手动分析的数字计时器,或用于自动分析的计算机软件(任意迷宫,Ethovision或其他) 注意:在我们的实验室,我们使用一个定制的加号迷宫大小为小鼠(Leo等人,2014),它是提高50厘米高于地面,包括两个相对的封闭的手臂,两个相反的开放臂和5厘米边的中心正方形(见下图)。闭合的手臂尺寸为30 x 5厘米,由白漆木制成,15厘米高的墙壁包围着手臂。应避免光泽绘画以防止过度眩光,因此建议使用哑光表面。开放臂尺寸为30 x 5厘米,由透明丙烯酸制成;一个0.3厘米高透明亚克力栏杆防止动物跌落,而探索开臂。该装置可以拆卸并移动到实验室,并从实验室移动到设置使用。木制装饰覆盖着木漆,漆成黑色或透明的有机玻璃装置也经常被发现。推荐使用市售的装置,例如来自Stoelting Co.或Panlab S.L.U.的装置作为替代品。房间照明和设备光反射强烈影响高架+迷宫测试。在我们的实验室,使用三星Flashcam数码摄像机,因为它更适合后期离线分析。在开始实验操作(药物,程序,转基因动物等)之前,应始终运行试验实验以建立可靠的基线参数并标准化实验环境。


    图1.正形迷宫

软件

  1. 自动计算机软件(任意迷宫,Ethovision等)

程序

  1. 迷宫应该安装在隔离的房间,远离任何噪音,气味或运动的任何外来干扰。 您可以选择在行为实验室中放置一个低强度白噪声源。 应当注意,指导提示,例如房间周围的壁上的绘图可能使动物的活动倾斜到迷宫的特定区域。 因此,任何可能充当线索的绘画或文物都必须删除 />
  2. 实验者必须约束在整个试验期间不产生任何过度的噪音或运动,以及佩戴香水,古龙水或任何具有强烈气味的产品,因为它可以作为小鼠的焦虑性刺激。
  3. 房间中的照明必须借助于勒克斯量表来测量,保持恒定并根据要进行的分析进行控制。由于低强度光度降低了开放臂避免(Morato和Castrechini,1989; Bertoglio和Carobrez,2002; Cosquer等人,2005; Pereira等人,2005年) ),为了分析焦虑效应,应该优选低强度照明(5-30Lux),而应当在较高强度照明(200-400Lux或更高)下分析抗焦虑效应。
  4. 在将这些实验条件调整到标准后,将动物带入实验室,在那里它们将在家笼中放置45至60分钟,以便从移动的应力中恢复。
  5. 在开始测试之前用70%乙醇清洁迷宫,以清除设备上积聚的任何污垢或气味
  6. 打开摄像机,将第一个鼠标放在迷宫的中心正方形,面向其中一个开放的臂,最好是与实验者相对的一个。
  7. 实验者将站在距离迷宫尽可能远的地方,并且在必要时在房间外面的测试动物的视野之外。他还必须避免不必要的运动或声音。
  8. 经过5分钟的自由探索,鼠标可能会移出迷宫,并回到其家笼。
  9. 所有的尿和粪便boli必须删除,迷宫用70%乙醇完全清除,以消除任何残留的气味从第一只老鼠。之后,下一个鼠标可能会提交测试。
  10. 重复步骤6-9,直到所有的动物都被测试
  11. 记录的视频可以通过自动计算机软件或者借助于计时器手动地分析。可以考虑几个参数:
    1. 闭合武器中的时间和在闭合武器中花费的时间:在整个实验期间对迷宫上的总运动的测量(Carola等人,2002; Walf和Frye,2007)。
    2. 在开放臂中的时间和在开放臂中花费的时间:可以用作焦虑的反向测量,也就是说,减少的开放臂避免反映较低的焦虑水平(Carola等人,2002; Walf和Frye,2007; Griebel等人,1993)。
    3. 风险评估行为:头部浸没的频率和持续时间(在开放臂上头部朝向地面向下运动)和伸展姿势(伸展姿势,头部和两个或三个爪在开放臂上,缩回到先前位置)可以是焦虑的直接测量,即增加的风险评估行为表示更高水平的焦虑(Carola等人,2002; Weiss等人, et al。,1998; Carobrez and Bertoglio,2005)
  12. 统计分析:应通过所有治疗方案的方差分析单独检查参数。如果适用,可以通过事后测试进一步分析数据。

代表数据



上图显示了我们设置中升高加迷宫(EPM)的典型数据,适用于调查焦虑反应(10 Lux)。对照组的行为是相对分布的,但是正确的统计学有效数据已经用每组6-8只小鼠的样品收集。药物1是一种清楚的抗焦虑药物,显示减少的开放臂时间和开放臂条目,没有改变所花费的时间和在闭合臂中的条目数量。药物2表明焦虑性样药理学特征的相对缺陷的解释,因为治疗的小鼠显示减少的运动,如通过每个臂中的条目的数量所揭示的。在这种情况下,我们建议药物剂量略有减少,以确定一个剂量,允许区分药物诱发的情绪和运动效应。还提供了我们在(Leo ,,2014)发表的一个实验的代表性视频。

笔记

  1. 通常,当进行行为实验时使用雄性小鼠,因为发情周期的变化通常影响雌性在这样的测试中的表现(Simpson等人,2012; da Silva等人 >,2014)。
  2. 基线活性可以根据小鼠品系,性别和年龄而变化(Leo等人,2014; Carobrez和Bertoglio,2005; Hogg,1996)。
  3. 动物应该被允许至少一周的习惯到家笼,因为他们往往在来自动物饲养设施到达时表现出高焦虑水平。
  4. 在我们的实验中,动物只处理笼清洁,称重,药物治疗和在测试期间。此外,动物只在光周期中测试;在黑暗循环中的测试可能产生不同的结果(Bertoglio和Carobrez,2002)
  5. 为了确保均匀的亮度,应始终使用间接光以避免产生硬阴影,这可能是小鼠偏好的地方,将其运动活动倾斜到迷宫的该区域。这可以通过适当的光反射器来完成
  6. 在测试每只动物后,应始终清洁迷宫。
  7. 确保清洁后迷宫完全干燥,或清洁剂的强烈气味可能改变小鼠的行为
  8. 如果在测试期间鼠标从开放臂脱落,实验者必须立即将其拾起并将其放回开放臂上。这在执行时应该注意和考虑 实验分析。该动物可以从最终分析中排除。
  9. 只有当动物将所有四只爪子从中心正方形中放到一个臂上时,才考虑打开或关闭臂中的入口。然而,给定手臂上的两只爪子已经足以停止计算进入手臂的时间。如果动物用这两只爪子回到手臂,计时器应该继续,但没有登记。只有在完全退出后才会注册新条目。
  10. 在高架加迷宫中没有行为测试的年龄相关限制的证据。尽管数据支持青少年和老年动物中增加的焦虑样状态(Lynn和Brown,2010; Bessa等人,2005; Chen等人,2007),但是,任务已经被证实用于成功评估青少年,成年,中年和老年啮齿类动物中的焦虑样行为(Leo等人,2014; Warneke等人 2014; Carrasco等人,2013; Joshi和Pratico,2011; Pietrelli等人,2012; Doremus等人,2006) 。

致谢

这里描述的协议已经改编自先前的研究(Leo等人,2014),其成功地鉴定小鼠的焦虑样样以及抗焦虑样行为。该项目得到了FAPERJ-Brasil(E-26/112.448/2012)和CNPq-Brasil(474443/2012-4)的财政支持。

参考文献

  1. Bertoglio,L.J。和Carobrez,A.P。(2002)。 在昼夜/夜间在高架加迷宫中提交给会话1 - 会话2的大鼠的行为概况阶段和在不同的照明条件下。 Behav Brain Res 132(2):135-143。
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引用:Leo, L. M. and Pamplona, F. A. (2014). Elevated Plus Maze Test to Assess Anxiety-like Behavior in the Mouse. Bio-protocol 4(16): e1211. DOI: 10.21769/BioProtoc.1211.
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