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Light/Dark Transition Test to Assess Anxiety-like Behavior in Mice
小鼠的类焦虑样行为的光照/黑暗转换试验   

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Abstract

The light/dark transition test (LDT) is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in response to mild stressors, such as novel environment and light. This test is also sensitive to anxiolytic drugs treatment. The test apparatus consists of a box divided into a small (one third) dark chamber and a large (two thirds) brightly illuminated chamber. Mice are placed into the lit compartment and allowed to move freely between the two chambers. The first latency to enter the dark compartment and the total time spent in lit compartment are indices for bright-space anxiety in mice. Transitions are index of activity-exploration, because of habituation over time. LDT is quick and easy to use, without requiring prior training of animals. Here, we present our protocol that has been able to detect both anxiolytic-like (reduced anxiety) and anxiogenic-like (increased anxiety) behavior in mice.

Keywords: Anxiety(焦虑), Light dark transition test(光暗转变试验), Behaviour(行为), Mouse(鼠标)

Materials and Reagents

  1. Paper towels
  2. Laboratory-bred mice
    Note: Mice housed in groups 3-5 per cage, kept in a room with controlled temperature (~23 °C) and humidity under 12 h light/dark cycle (lights on at 7:00 AM) with ad libitum access to food and water.
  3. 70% ethanol

Equipment

  1. The apparatus for the light/dark transition test consists of a box (42 x 21 x 25 cm) divided into a small (one third) dark compartment and a large (two thirds) illuminated compartment (Crawley and Goodwin, 1980). A restricted opening 3 cm high by 4 cm wide connects the two chambers (Figure 1).
  2. Indirect white light source
  3. Digital lux meter
  4. Video camera (placed directly above the apparatus)
  5. Digital chronometers for manual analysis or computer for automated analysis


    Figure 1. Light-dark transition test apparatus

Software

  1. TSE-Systems, model: VideoMot2 (computer software for automated analysis)

Procedure

  1. The apparatus is placed in an isolated room away from any external interferences and noises with a low-intensity white light source.
  2. The experimenter must restrict from making any excessive noise during the test and from wearing odorant products with strong smell, since it could act as an anxiogenic stimulus for the mice.
  3. The illumination in the lit chamber must be 200-400 lux or more, while the other chamber is dark (5 lux or less).
  4. The mice are transferred in their home cages to the behavioral testing room for at least 30 min before the experiment.
  5. Clean both compartments of the apparatus with 70% ethanol.
  6. Turn on the camera and place the first mouse in the middle of the brightly illuminated chamber.
  7. The experimenter stays as far away as possible from the box and out of sight of the test animal.
  8. The mouse is allowed to move freely between the two chambers for 5 min (Costall et al., 1989).
  9. After each trial, all the urine and fecal boli are removed and both chambers are cleaned with 70% ethanol.
  10. The recorded videos can be analyzed by automated computer software or manually with the aid of chronometer. The following parameters are scored (Figure 2):
    1. The latency time to enter the dark compartment (Costall et al., 1989).
    2. The time spent in the lit/dark compartment.
    3. The distance travelled in the lit chamber.
    4. The number of transitions.


Representative data



Figure 2. Sample data from light-dark transition test showing anxiolytic-like behavior of TG mice. A. Time spent in the lit compartment; B. Traveled distance in the lit compartment; C. latency time to enter the dark compartment; D. Number of transitions.

Notes

  1. The LDT was originally developed by Crawley and colleagues (Crawley and Goodwin, 1980). There are two differences between the original version and our test. First, the duration of the test is 10 min in the original version, whereas we used only 5 min testing time (Costall et al., 1989). Second, in the original version, the walls of the lit compartment are transparent, while we use opaque white plastic for the walls. Thus our test protocol is more specific for detecting bright-space anxiety, compared to the original version, where also open-space anxiety is scored.
  2. Typically, male mice are used for behavioral experiments, as variations of estrous cycle might influence the performance of females in such tests (Simpson et al., 2012).
  3. Baseline activity may vary according to mouse strain and age.
  4. In order to ensure uniform luminosity, indirect light is used to avoid the production of shadows, which can be a place of preference for the mouse.
  5. The both compartments must always be cleaned after the testing of each animal and fully dried after cleaning, because the strong smell of the cleaning agent might alter the mouse behavior.
  6. An entry in to the lit or dark compartment is only considered when the animal places 2 frontal paws inside it. If the animal return with these two paws into the compartment no entry is registered.

Acknowledgments

The protocol described here has been adapted from a previous study (Serchov et al., 2015), which succeeded in identifying anxiolytic-like behavior for mice. This work was supported by grants from the German Research Council (DFG) (CA 115/5-4) to D.v.C. and K.B., the European Union FP7 program “MoodInflame” to D.v.C. and German Ministry for Research and Education (DMBF) grant e:bio-Modul I -ReelinSys (Project B: 031 6174A) to K.B.

References

  1. Costall, B., Jones, B. J., Kelly, M. E., Naylor, R. J. and Tomkins, D. M. (1989). Exploration of mice in a black and white test box - validation as a model of anxiety. Pharmacol Biochem Behav 32(3): 777-785.
  2. Crawley, J. and Goodwin, F. K. (1980). Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 13(2): 167-170.
  3. Serchov, T., Clement, H. W., Schwarz, M. K., Iasevoli, F., Tosh, D. K., Idzko, M., Jacobson, K. A., de Bartolomeis, A., Normann, C., Biber, K. and van Calker, D. (2015). Increased signaling via adenosine A1 receptors, sleep deprivation, imipramine, and ketamine inhibit depressive-like behavior via induction of Homer1a. Neuron 87(3): 549-562.
  4. 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.

简介

光/暗转变测试(LDT)是最广泛使用的测试小鼠焦虑样行为的测试之一。该测试基于小鼠对明亮照明区域的自然厌恶以及其对轻度应激物例如新环境和光的响应的自发探索行为。该试验也对抗焦虑药物治疗敏感。测试装置由分成小(三分之一)暗室和大(三分之二)明亮照明室的盒子组成。将小鼠放入点燃的隔室中并允许在两个室之间自由移动。进入暗室的第一个等待时间和光照隔室中花费的总时间是小鼠明亮焦虑的指标。过渡是活动探索的指数,因为随着时间的习惯。 LDT快速和容易使用,无需事先训练动物。在这里,我们提出我们的协议,已经能够检测小鼠抗焦虑样(减少焦虑)和焦虑类似(焦虑)行为。

关键字:焦虑, 光暗转变试验, 行为, 鼠标

材料和试剂

  1. 纸毛巾
  2. 实验室饲养的小鼠
    注意:将小鼠保持在每组3-5只,保持在具有控制温度(?23℃)和湿度的房间中,12小时光照/黑暗循环(上午7:00点亮),随意进入到食物和水。
  3. 70%乙醇

设备

  1. 用于明/暗转换测试的装置由分为小(三分之一)暗室和大(三分之二)发光隔室(Crawley和Goodwin,1980)的盒子(42×21×25cm)组成。 3厘米高,4厘米宽的限制开口连接两个室(图1)
  2. 间接白光源
  3. 数字照度计
  4. 摄像机(直接放在设备上方)
  5. 用于手动分析的数字计时器或用于自动分析的计算机


    图1.光暗转变测试仪

软件

  1. TSE-Systems,型号:VideoMot2(用于自动分析的计算机软件)

程序

  1. 该设备放置在隔离的房间中,远离任何外部干扰和低强度白光源的噪声
  2. 实验者必须限制在测试期间产生任何过度的噪声和具有强烈气味的气味产品的佩戴,因为它可以作为小鼠的抗焦虑刺激。
  3. 在照明室内的照明必须是200-400勒克斯或更多,而另一个室是黑暗(5勒克斯或更少)。
  4. 在实验前将小鼠在家笼中转移到行为测试室至少30分钟
  5. 用70%乙醇清洁设备的两个隔室
  6. 打开相机,将第一个鼠标放在明亮照明的房间中间。
  7. 实验者尽可能远离箱子,并且远离测试动物的视线。
  8. 允许小鼠在两个室之间自由移动5分钟(Costall等人,1989)。
  9. 每次试验后,取出所有的尿和粪便,并用70%乙醇清洗两个室
  10. 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。对以下参数进行评分(图2):
    1. 进入暗室的延迟时间(Costall等人,1989)。
    2. 在照明/暗室中的时间
    3. 在照明室内行进的距离。
    4. 转换次数。


代表数据



图2.来自光 - 暗转换测试的样品数据显示TG小鼠的抗焦虑样行为。 A.在光照室中花费的时间; B.在照明室内行驶的距离; C.进入暗室的延迟时间; D.转换数。

笔记

  1. LDT最初由Crawley及其同事开发(Crawley和Goodwin,1980)。在原始版本和我们的测试之间有两个区别。首先,在原始版本中,测试的持续时间为10分钟,而我们仅使用5分钟测试时间(Costall等人,1989)。第二,在原来的版本中,照明隔间的墙是透明的,而我们使用不透明的白色塑料的墙壁。因此,与原始版本相比,我们的测试协议对于检测明亮空间焦虑更具体,其中也开放空间焦虑得分。
  2. 通常,雄性小鼠用于行为实验,因为发情周期的变化可能影响雌性在这种测试中的表现(Simpson等人,2012)。
  3. 基线活性可根据小鼠品系和年龄而变化
  4. 为了确保均匀的亮度,使用间接光以避免产生阴影,这可以是鼠标的优选地点。
  5. 两个隔室必须在每个动物测试后始终清洁,并在清洁后完全干燥,因为清洁剂的强烈气味可能改变小鼠的行为。
  6. 只有当动物在其中放置2只额爪时,才考虑进入亮的或暗的隔间。如果动物用这两只爪子回到隔间,则没有登记入口

致谢

这里描述的协议已经改编自以前的研究(Serchov等人,2015),成功地识别小鼠的抗焦虑样行为。这项工作是由德国研究委员会(DFG)(CA 115/5-4)授予D.v.C。和K.B.,欧盟FP7程序"MoodInflame"至D.v.C.和德国研究和教育部(DMBF)给予K.B.的生物模拟I-ReelinSys(项目B:031 6174A)。

参考文献

  1. Costall,B.,Jones,BJ,Kelly,ME,Naylor,RJ和Tomkins,DM(1989)。  在黑色和白色试验箱中探测小鼠 - 作为焦虑模型的验证药物生物化学 :777-785。
  2. Crawley,J.和Goodwin,FK(1980)。  苯并二氮杂卓的抗焦虑作用的简单动物行为模型的初步报告.Pharmacol Biochem Behav 13(2):167-170。
  3. Serchov,T.,Clement,HW,Schwarz,MK,Iasevoli,F.,Tosh,DK,Idzko,M.,Jacobson,KA,de Bartolomeis,A.,Normann,C.,Biber,K.and van Calker, D.(2015)。  通过腺苷A1受体增加信号传导,睡眠剥夺,丙咪嗪和氯胺酮通过诱导Homer1a抑制抑郁样行为。 Neuron 87(3):549-562。
  4. Simpson,J.,Ryan,C.,Curley,A.,Mulcaire,J.和Kelly,JP(2012)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Serchov, T., van Calker, D. and Biber, K. (2016). Light/Dark Transition Test to Assess Anxiety-like Behavior in Mice. Bio-protocol 6(19): e1957. DOI: 10.21769/BioProtoc.1957.
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