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Jul 2017

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Classic Labyrinth Test for Neurobehavioral Evaluation in Wistar Rats
利用经典迷宫实验对大鼠进行神经行为功能评价   

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Abstract

The Classic Labyrinth Test (CLT) is a simple way to evaluate behaviors in rodents such as learning ability, memory, and anxiety. The protocol presented here describes the procedure for use with rats, but the protocol can also be adapted for use in mice if a smaller device is used. In short, the CLT uses a square-shaped maze with a starting point and a stopping point. After the animal is trained, the animal is allowed to view and explore the labyrinth freely for 10 min. During this time, all of the animal's vertical and horizontal movements within the labyrinth are recorded. This is a very challenging task because it requires the animal to remember the quickest path between the starting points and the end. In cases where the labyrinth is designed so that the animal only needs to walk forward, it is quite easy for healthy rats, but for rats exposed to neuro-xenobiotics (drugs, pesticides) there will be disturbances in their path. Researchers use many different versions of this test and the procedure for each version can vary significantly. Here, we present a working protocol that enables the detection of traces of some toxic substances that may be exposed to individuals over a long period and in very small amounts under specific conditions such as drugs, medicines and pesticides.

Keywords: Classic labyrinth (经典迷宫测试), Behavior (行为), Learning (学习), Drugs (药物), Pesticides (杀虫剂), Rats (大鼠)

Background

We set out to elucidate how animals memorize and learn when they are challenged with stress utilizing the classical labyrinth test. The CLT is performed in a square-shaped plastic enclosure (125 x 125 x 40 cm) with several labyrinth passages of identical width and height (25 x 35 cm), but with variable length (Figure 1). Usually, this labyrinth is placed on a table 90 cm high. Control rats can quickly move through the labyrinth between the starting point and the ending point. When the animal explores the maze, it increases the time it spends in the maze's passages, which will be considered as aversive or anxiety-provoking for the stressed animal; while the leak behavior will be observed when the animal spends more time in the starting point or corners, which will be associated with a refuge (Leo and Pamplona, 2014; Gasmi et al., 2017b). There are many tests similar to this test, but this test is unique because it is concerned with measuring the impact of environmental pollutants on the behavior and psychology of animals, especially rats (Serchov et al., 2016). Some advantages of this approach are that this test is inexpensive, and it is quick; the test usually takes about 10 min, and after cleaning the labyrinth with 70% ethanol, it can be used to test the next animal (Gasmi et al., 2017a and 2018).

Materials and Reagents

  1. Paper towels
  2. Laboratory rats (3 months old and weight between 200 and 240 g)
    The rats are housed in groups of 4 per cage and kept in an environment with temperature (23 ± 2 °C) and humidity control. The animals have a 12-12 h light-dark cycle and food and water ad libitum.
  3. Ethanol (70%) for cleaning (CID: 702)
  4. Plastic sheets (PVC wall panel) (Dacheng, model: DCb-008 )
  5. Adhesive glue for plastics (SG300-05)

Equipment

  1. Home cage (Tecniplast, catalog number: 1291H001, EUROSTANDARD TYPE III H, 425 x 266 x 185 mm)
  2. Classic Labyrinth for rats [a square shaped plastic enclosure (125 x 125 x 40 cm) with several labyrinth passages of identical width and height (25 x 35 cm) but variable length according to the crossing. This Labyrinth can be assembled manually as shown in Figure 1, where it is made of white plastic sheets. These plates are smooth and solid and should be easy to clean and unbreakable or damaged by the animal. The maze is formed at 125 x 125 x 40 cm by welding the plastic sheets together with adhesive glue. According to Figure 1, after the maze is made, it is placed on a wooden table 90 cm high on the ground (Figure 1)]
  3. USB Camera Lenses (ITEM: 60528/ Lens for 60516, Vari-focal, 2.8-12 mm)
    Note: It was placed directly in the middle above the Labyrinth at the height of 1.20 m so that only the labyrinth was exposed.
  4. Chronometer (LCD Digital Portátil Reloj Alarma Temporizador by SYMTOP)


    Figure 1. Classic Labyrinth and its dimensions

Software

  1. Ethovision video tracking system software (version 10, Noldus Information Technology, France)
  2. Microsoft Excel (Microsoft Corp® 2016, USA)
  3. Minitab (Minitab® version 17.1, USA)

Procedure

  1. Several exercises are performed for the rat (about four to five times as needed) before the final data are recorded, 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 (Leo et al., 2014), During the training period, rewards with an odor cue (bread, grease, cheese, processed food, etc.) are used to attract the rats and help them quickly reach the end of the maze (the target). However, on the test day, nothing is left at leaves nothing at the stopping point. We do this because it eliminates external factors and variables (e.g., odor) between experiments (Gasmi et al., 2017b).
  2. The apparatus (labyrinth) is placed in an isolated room away from any external interferences and noises with a low-intensity white light source.
  3. The experimenter must avoid making any excessive noises during the test and from wearing products with a strong smell because it could act as an anxiogenic stimulus for the mice.
  4. The illumination in the lit chamber must be 200-400 lux.
  5. The rats are transferred in their home cages to the behavioral testing room for at least 60 min before the experiment.
  6. Clean both compartments of the apparatus with 70% ethanol (Serchov et al., 2016).
  7. Turn on the camera and place the first rat in the starting point of labyrinth.
  8. The experimenter stays as far away as possible from the box and out of sight of the test animal.
  9. The rats are allowed to move freely within the labyrinth for 10 min (between the starting point and the stopping), but in exceptional cases 10 min is added to the test for a total of 20 min. 
  10. After each trial, all urine and fecal matter are removed, and the maze is cleaned with 70% ethanol.
  11. The recorded videos can be analyzed by automated computer software (Ethovision video tracking system software, Spink et al., 2001) or manually with the aid of a chronometer.
  12. The duration of time required for the animal to move from the starting point to the stopping point (Arrival Time) is recorded (Figure 2). If the animal takes significantly longer to arrive at the stopping point, this delay might be explained by external factors that lead to decreased cognition. The arrival time is determined by when the rat reaches the stop point and then stays there for at least 10 sec.


    Figure 2. Representative data (mean ± SD) of the Arrival Time of control rats in the classical labyrinth test (C) and rats treated with a mixture of pesticides (D+A)

Data analysis

The results obtained were expressed as the average of six repetitions (mean ± standard deviation), and the data were visualized using Excel 2013 to represent these results in the form of graphs and histograms. Statistical analysis was performed using the Minitab® 17.1 software. The significance of difference between the control and the treated lots is verified using the Student’s t-test, and the comparison result as follows:
P > 0.05 = The difference is not significant.
(*) 0.05 > P > 0.01 = The difference is significant.
(**) 0.01 > P > 0.001 = The difference is highly significant.
(***) P < 0.001 = The difference is very highly significant.

Notes

  1. Testing this maze is one of the easiest and most effective tests.
  2. Experiment does not consume a lot of equipment, which makes it cost effective.
  3. We have used this test in our laboratory to study the negative impact of many chemicals on the neurological health of animals.
  4. There can be a significant difference between the sexes. We recommend using male or female rats only. We do not recommend using both sexes in one test.
  5. Failure to clean the maze well between tests and using a poorly prepared room can lead to bad/unreliable results. 
  6. Mice can be used in this type of test (maze test). However, in this protocol only the Wister rats were used. To evaluate if a rat is ready for the final test, we expect the rat to pass all of the initial training classes during the first five days of training. 
  7. Generally, over-training is not harmful and may last up to five days, but all groups of rats must undergo the same experimental conditions and the same duration of training. As any difference in the duration or number of training sessions will have a negative impact on the results of the test.
  8. If the rat reaches the stop point and immediately departs (10 sec before it stops), do not record the result. The experiment will continue normally and the arrival time will be recorded only when the rat stops more than 10 sec at the stop. If the first ten minutes are completed without the rat reaching the breakpoint, another 10 min of the test will be added and the entire period (20 min) will be counted.
  9. The average duration of the experiment is 10 min. If the animal does not reach the stopping point, another 10 min will be added. The total possible time is 20 min. When the rat reaches the stop point during this period, the test is successful. If the rat does not reach the stopped point during this period (20 min), the test is considered to be a failure (the test failed) and is returned after three hours.
  10. A simple and possible analysis has been provided by hand. If the Ethovision video tracking system software is used to analyze and present the results such as learning curve, error rate, memory ability, neurological state, etc.
  11. Daily training of the rats for four or five days will help them remember the shortest path in the maze to reach the stopping point, but when these animals are exposed to neurotoxic drugs or environmental pollutants it becomes difficult for them to identify the right path in the Labyrinth.
  12. In this study, the statistical test "t" is indicated because it is the basic test that the novice researcher can perform, but this does not preclude the use of the other types of statistical tests according to the type of study and the researcher's need. As for the results presented, it is the mean + SD.

Acknowledgments

This protocol has been used in our published works (Gasmi et al., 2017b and 2018).
This research did not receive any specific grant from funding agencies in the public, commercial, or not for profit sectors. Many thanks to Pr. Kebieche M, and Pr. Rouabhi R and Benkhedir AK. Also to Dr. Boussekine S chef of applied biology department, University of Tebessa for providing advice and guidance.

Competing interests

I declare there are no competing interests.

Ethics

This study was conducted in accordance with the EU Commission Directive 2010/63/EU for animal experiments, after approval of the Scientific Committee of Applied Biology Faculty - University of Tebessa, which issued a permit No. 247SNV/12/2014.

References

  1. Gasmi, S., Kebieche, M., Rouabhi, R., Touahria, C., Lahouel, A., Lakroun, Z., Henine, S. and Soulimani, R. (2017a). Alteration of membrane integrity and respiratory function of brain mitochondria in the rats chronically exposed to a low dose of acetamiprid. Environ Sci Pollut Res Int 24(28): 22258-22264.
  2. Gasmi, S., Rouabhi, R., Kebieche, M., Boussekine, S., Salmi, A., Toualbia, N., Taib, C., Bouteraa, Z., Chenikher, H., Henine, S. and Djabri, B. (2017b). Effects of Deltamethrin on striatum and hippocampus mitochondrial integrity and the protective role of Quercetin in rats. Environ Sci Pollut Res Int 24(19): 16440-16457.
  3. Gasmi, S., Rouabhi, R., Kebieche, M. and Menaceur, F. (2018). Neurotoxicité des pesticides chez les rats. EUE 224.
  4. 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.
  5. 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.
  6. Spink, A. J., Tegelenbosch, R. A., Buma, M. O. and Noldus, L. P. (2001). The EthoVision video tracking system-a tool for behavioral phenotyping of transgenic mice. Physiol Behav 73(5):731-744.

简介

经典迷宫测试(CLT)是一种评估啮齿动物行为的简单方法,如学习能力,记忆力和焦虑。此处介绍的方案描述了与大鼠一起使用的程序,但如果使用较小的装置,该方案也可适用于小鼠。简而言之,CLT使用具有起点和停止点的方形迷宫。在训练动物后,允许动物自由观察和探索迷宫10分钟。在此期间,记录迷宫内所有动物的垂直和水平运动。这是一项非常具有挑战性的任务,因为它需要动物记住起点和终点之间的最快路径。如果迷宫的设计使得动物只需要向前走,那么健康的老鼠就很容易,但是对于暴露于神经 - 异生素(药物,杀虫剂)的大鼠来说,它们的路径会受到干扰。研究人员使用此测试的许多不同版本,每个版本的过程可能会有很大差异。在这里,我们提出了一个工作方案,能够检测某些有毒物质的痕迹,这些物质可能在很长一段时间内暴露给个人,并且在特定条件下,如药物,药物和杀虫剂,可能会非常少量。

【背景】我们着手阐明动物在利用经典迷宫测试受到压力挑战时如何记忆和学习。 CLT在方形塑料外壳(125 x 125 x 40 cm)中进行,带有几个相同宽度和高度(25 x 35 cm)的迷宫通道,但长度可变(图1)。通常,这个迷宫放在90厘米高的桌子上。对照组大鼠可以在起点和终点之间快速穿过迷宫。当动物探索迷宫时,它增加了它在迷宫通道中花费的时间,这将被视为对压力动物的厌恶或焦虑。当动物在起点或角落花费更多时间时将观察泄漏行为,这将与避难所相关联(Leo和Pamplona,2014; Gasmi et al。,2017b)。有许多类似于这个测试的测试,但这个测试是独一无二的,因为它涉及测量环境污染物对动物,特别是大鼠的行为和心理的影响(Serchov et al。,2016) 。这种方法的一些优点是该测试价格低廉,而且速度快;测试通常需要大约10分钟,在用70%乙醇清洗迷宫后,它可以用来测试下一个动物(Gasmi et al。,2017a和2018)。

关键字:经典迷宫测试, 行为, 学习, 药物, 杀虫剂, 大鼠

材料和试剂

  1. 纸巾
  2. 实验室大鼠(3个月大,体重在200到240克之间)
    将大鼠圈养在每笼4只的组中,并保持在温度(23±2℃)和湿度控制的环境中。这些动物具有12-12小时的明暗循环和随意的食物和水。
  3. 乙醇(70%)用于清洁(CID:702)
  4. 塑料板(PVC墙板)(大成,型号:DCb-008)
  5. 塑料用胶水(SG300-05)

设备

  1. 家用保持架(Tecniplast,目录号:1291H001,EUROSTANDARD TYPE III H,425 x 266 x 185 mm)
  2. 用于老鼠的经典迷宫[方形塑料外壳(125 x 125 x 40 cm),带有几个相同宽度和高度(25 x 35 cm)的迷宫通道,但根据交叉点可变长度。这个迷宫可以手动组装,如图1所示,它由白色塑料板制成。这些板是光滑和坚固的,应该易于清洁,不易被动物破坏或损坏。通过用胶粘剂将塑料片焊接在一起,形成125×125×40cm的迷宫。根据图1,在制作迷宫后,将其放置在地面上90厘米高的木桌上(图1)]
  3. USB相机镜头(ITEM:60528 /镜头适用于60516,可变焦距,2.8-12 mm)
    注意:它直接放在迷宫上方1.20米高处的中间位置,只露出迷宫。
  4. 天文台(SYMTOP的LCDDigitalPortátilRelojAlarma Temporizador)


    图1.经典迷宫及其尺寸

软件

  1. Ethovision视频跟踪系统软件(第10版,Noldus信息技术,法国)
  2. Microsoft Excel(Microsoft Corp ® 2016,USA)
  3. Minitab(Minitab ®版本17.1,美国)

程序

  1. 在记录最终数据之前,对大鼠进行几次锻炼(大约需要四到五次),动物应该被允许至少一周适应家庭笼子,因为他们在抵达时往往会出现高焦虑水平。动物饲养设施(Leo et al。,2014),在培训期间,带有气味提示(面包,油脂,奶酪,加工食品,等)的奖励是用来吸引老鼠并帮助他们快速到达迷宫的末端(目标)。然而,在测试当天,没有任何东西留在停止点没有任何东西。我们这样做是因为它消除了实验之间的外部因素和变量(例如,气味)(Gasmi et al。,2017b)。
  2. 将设备(迷宫)放置在隔离的房间中,远离任何外部干扰和噪声,使用低强度白光源。
  3. 实验者必须避免在测试过程中产生任何过度噪音,并佩戴具有强烈气味的产品,因为它可以作为小鼠的焦虑刺激物。
  4. 点亮室内的照度必须为200-400勒克斯。
  5. 在实验前将大鼠在其家笼中转移至行为测试室至少60分钟。
  6. 用70%乙醇清洁设备的两个隔室(Serchov 等人,2016)。
  7. 打开相机,将第一只老鼠放在迷宫的起点。
  8. 实验者尽可能远离盒子并且看不到测试动物。
  9. 允许大鼠在迷宫内自由移动10分钟(在起始点和停止之间),但在特殊情况下,在测试中加入10分钟,总共20分钟。&nbsp;
  10. 每次试验后,除去所有尿液和粪便物质,用70%乙醇清洗迷宫。
  11. 录制的视频可以通过自动计算机软件(Ethovision视频跟踪系统软件,Spink et al。,2001)进行分析,也可以借助计时器手动进行分析。
  12. 记录动物从起始点移动到停止点(到达时间)所需的持续时间(图2)。如果动物需要更长的时间才能到达停止点,这种延迟可能是由导致认知能力下降的外部因素造成的。到达时间取决于大鼠何时到达停止点然后在那里停留至少10秒。


    图2.经典迷宫试验( C )和用杀虫剂混合物处理的大鼠( D + A)的对照大鼠的到达时间的代表性数据(平均值±SD) )

数据分析

获得的结果表示为六次重复的平均值(平均值±标准偏差),并且使用Excel 2013将数据可视化以图表和直方图的形式表示这些结果。使用Minitab ® 17.1软件进行统计学分析。使用Student's t -test验证对照和处理批次之间差异的显着性,比较结果如下:
P &gt; 0.05 =差异不显着。
(*)0.05> P &gt; 0.01 =差异很大。
(**)0.01> P &gt; 0.001 =差异非常显着。
(***) P &lt; 0.001 =差异非常显着。

笔记

  1. 测试这个迷宫是最简单和最有效的测试之一。
  2. 实验不会消耗大量设备,因此具有成本效益。
  3. 我们在实验室中使用该测试来研究许多化学物质对动物神经健康的负面影响。
  4. 两性之间可能存在显着差异。我们建议仅使用雄性或雌性大鼠。我们不建议在一次测试中使用两种性别。
  5. 未能在测试之间清洁迷宫并使用准备不足的房间会导致不良/不可靠的结果。&nbsp;
  6. 小鼠可以用于这种类型的测试(迷宫测试)。然而,在该方案中仅使用 Wister 大鼠。为了评估大鼠是否准备好进行最终测试,我们希望大鼠在培训的前五天通过所有初始培训课程。&nbsp;
  7. 一般来说,过度训练是无害的,并且可能持续长达五天,但是所有组的大鼠必须经历相同的实验条件和相同的训练持续时间。由于培训课程的持续时间或数量的任何差异都会对测试结果产生负面影响。
  8. 如果老鼠到达停止点并立即离开(停止前10秒),请不要记录结果。实验将继续正常进行,只有当大鼠在停止时停止超过10秒时才会记录到达时间。如果在没有大鼠到达断点的情况下完成前十分钟,则将添加另外10分钟的测试并且将计算整个时段(20分钟)。
  9. 实验的平均持续时间为10分钟。如果动物未到达停止点,则再加入10分钟。总可能的时间是20分钟。当大鼠在此期间到达停止点时,测试成功。如果大鼠在此期间(20分钟)没有达到停止点,则认为测试失败(测试失败)并在3小时后返回。
  10. 手工提供了简单而可能的分析。如果Ethovision视频跟踪系统软件用于分析和呈现结果如学习曲线,错误率,记忆能力,神经状态,等
  11. 每天训练大鼠四到五天将帮助他们记住迷宫中最短的路径到达停止点,但当这些动物暴露于神经毒性药物或环境污染物时,他们很难确定正确的路径。迷宫。
  12. 在本研究中,指出统计检验“t”是因为它是新手研究人员可以执行的基本测试,但这并不排除根据研究类型和研究人员的需要使用其他类型的统计检验。至于呈现的结果,它是平均值+ SD。

致谢

该协议已经在我们的出版的作品被使用(Gasmi 等人,2017b和2018)。
该研究没有从公共,商业或非营利部门的资助机构获得任何特定资助。非常感谢Pr。 Kebieche M和Pr。 Rouabhi R和Benkhedir AK。同时为Tebessa大学应用生物学系的Boussekine博士提供建议和指导。

利益争夺

我们宣布没有竞争利益。

伦理

本研究是根据欧盟委员会指令2010/63 / EU进行的动物实验,经Tebessa大学应用生物学科学委员会批准,该委员会颁发了第247SNV / 12/2014号许可证。

参考

  1. Gasmi,S.,Kebieche,M.,Rouabhi,R.,Touahria,C.,Lahouel,A.,Lakroun,Z.,Henine,S。和Soulimani,R。(2017a)。 长期暴露于低剂量啶虫脒的大鼠脑线粒体膜完整性和呼吸功能的改变。 Environ Sci Pollut Res Int 24(28):22258-22264。
  2. Gasmi,S.,Rouabhi,R.,Kebieche,M.,Boussekine,S.,Salmi,A.,Toualbia,N.,Taib,C.,Bouteraa,Z.,Chenikher,H.,Henine,S。and Djabri,B。(2017b)。 溴氰菊酯对纹状体和海马线粒体完整性的影响以及槲皮素对大鼠的保护作用。 Environ Sci Pollut Res Int 24(19):16440-16457。
  3. Gasmi,S.,Rouabhi,R.,Kebieche,M。和Menaceur,F。(2018)。 Neurotoxicit des des pesticides chez les rats。 EUE 224。
  4. Leo,L.M和Pamplona,F.A。(2014)。 升高加迷宫测试以评估鼠标中的焦虑行为。 生物协议 4(16):e1211。
  5. Serchov,T.,van Calker,D。和Biber,K。(2016)。 光/暗过渡测试,以评估小鼠的焦虑行为。 生物协议 6(19):e1957。
  6. Spink,A.J.,Tegelenbosch,R.A.,Buma,M.O。和Noldus,L。P.(2001)。 EthoVision视频跟踪系统 - 转基因小鼠行为表型的工具。 Physiol Behav 73(5):731-744。
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引用:Gasmi, S. (2018). Classic Labyrinth Test for Neurobehavioral Evaluation in Wistar Rats. Bio-protocol 8(18): e3007. DOI: 10.21769/BioProtoc.3007.
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