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Measuring Anxiety-like Behavior in Crayfish by Using a Sub Aquatic Dark-light Plus Maze
使用水下暗光十字迷宫法测试小龙虾的焦虑样行为   

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Abstract

Crayfish are omnivorous freshwater arthropods that naturally explore their environment during day and night, but also frequently hide under a shelter or in a hole in case of danger. They may be submitted to various stressors, including predation, social interactions or changes in environmental parameters (temperature, water quality, oxygen, etc.). It has been recently demonstrated that, as a consequence of stress, crayfish is able to adapt its exploratory behavior by restricting movements to protective areas, a response similar to the anxiety-like behavior (ALB) observed in rodents. To reveal such a behavior in an aquatic species, we designed a plus-shaped sub aquatic maze divided in two protective dark arms and two more aversive illuminated arms. The aim of this paradigm was to place crayfish in a conflicting situation between its innate curiosity for novel environment and its aversion for light (Leo, 2014; Pellow et al., 1985). Unstressed crayfish generally explore the whole maze, including illuminated arms. By contrast stressed crayfish remain preferentially in the dark arms (Fossat et al., 2014). Stressed crayfish injected with anxiolitics (chlordiazepoxide-CDZ), behave as unstressed animals. Several parameters, related to the light arms can be easily measured from video records by commercial software This protocol could be suitable for analyzing the effects of any stressful situation on ALB in crayfish, as well as in many other aquatic species.

Materials and Reagents

  1. Animals
    1. The study has been performed on adult male crayfish, Procambarus clarkii , fished near Bordeaux (France) in the "Réserve naturelle de Bruges" and stored in an animal house with 12:12 dark light cycle. Crayfish were fed ad libitum with pellets (Novo Prawn). Crayfish used for this study were 8.7 ± 0.8 cm and 22.1 ± 1.1 g. We generally used male crayfish in our experiments (distinguished by their first abdominal appendages), but this protocol is also suitable for experiments on females.
    2. Before any experiment, crayfish were isolated in enriched aquariums (75 cm x 40 cm x 50 cm), filled with recycled oxygenated water and enriched with pebbles and plastic tubes (behind or inside which they can hide). Animals were kept isolated at least 3 weeks before any experiment in order to erase past life histories and to avoid social interactions.
    3. Water must be chlorine free (agitating fresh tap water during 24 h is generally enough to remove chlorine). Change water after each session or use a conventional filtering system for aquarium.
    4. Fishnets are used to manipulate crayfish.

  2. Reagents
    1. Ringer: Crayfish were injected with a classical invertebrate ringer containing 195 NaCl, 5 KCl, 13 CaCl2, 2 MgCl2 and 3 HEPES (Sigma-Aldrich) with a pH of 7.65
    2. Chlordiazepoxide hydrochloride (CDZ) (Sigma-Aldrich) (dissolved in crayfish ringer and was used at a dose 15 µg/g)

Equipment

  1. Dark/light (D/L) plus maze
    1. Customized apparatus (65 x 65 cm, see Figure 1) is adapted to the size of adult crayfish. Smaller or larger dimensions might be necessary for young crayfish or other species.
    2. The illuminated arms were made of transparent Plexiglas and dark arms of opaque Plexiglas (Figure 1).
    3. The maze is filled with ca. 7 cm of oxygenated freshwater. It is placed in the center of a larger round tank (Figure 2). The round tank (1.5 m in diameter) is also filled with 7 cm freshwater to equilibrate water pressure on each maze wall and to allow for good oxygenation and recycling of water (Figure 2).


      Figure 1. Dark/light plus maze. The total length of the maze is 65 cm x 65 cm. Each arm is 25 cm long and 15 cm wide. The middle zone is 15 cm x 15 cm. The light is generated by four sources comprising two LEDs. Each source of light is placed on the outside face of the opaque Plexiglas.


      Figure 2. The dark/light plus maze is placed in a larger tank (diameter 1.5 m). Right: Room light switch on. Left: Room light switch off under experimental conditions.

    4. Illumination of the light arms is performed by a series of LEDs (white light; voltage; 3.5 V, light intensity; 880 to 2,530 mcd) disposed outside the plus maze, on the outward side of the dark arms on the outmost extremities (see Figure 3).


      Figure 3. Two LEDs placed at the end of the dark arm illuminate a light arm. The height of the maze wall is 20 cm.

  2. Other devices
    1. A video camera must be placed above the maze. We used a handycam (Sony inc, model: HDR-XR155) but any commercial camera can be used.
    2. A pump (NewJet) is placed on a large tank between experimental sessions to agitate and oxygenate water.
    3. Light intensity was assessed with a luxmeter (Chauvin Arnoux).

Software

  1. We used Ethovision v8 (Noldus) but any tracking software can be used. Measured parameters are listed in Table 1.

    Table 1. List of parameters that can be used to analyse the anxiety-like behaviour in the D/L plus maze
    Name of variable (and abbreviation used in Figure 2)
    Definition
    Measurement
    Range
    Total distance moved (Distance)
    Distance moved in the entire arena.
    cm
    ≥0
    % Time in zone (%T light, dark)
    Time spent in a zone (dark or light)/(divided by total time)*100
    %
    0-100
    Latency to first in light (Lat.Light)
    Time from start time to first entry in a light arm
    Second (s)
    0-600
    Latency to first in dark (Lat.Dark)
    Time from start time to first entry in a dark arm
    Second (s)
    0-600
    Mean number of entry in light arms (Nb. ent. Dark)
    Number of entry from middle zone to a light arm
    NA
    ≥0
    Mean number of entry in dark arms (Nb. ent. Light)
    Number of entry from middle zone to a dark arm
    NA
    ≥0
    Mean duration per visit in light arms
    Time in light arms/mean number of entry
    Second (s)
    ≥0
    Mean duration per visit in dark arms
    Time in dark arms/mean number of entry
    Second (s)
    ≥0
    Retreat Ratio (RR)
    Number of retreat / total number of attempts
    NA
    0-1

Procedure

  1. Because our maze has been developed to measure the consequences of stress, crayfish must be submitted to some stressful situation before being tested in the dark/light plus maze. Any source of stress can be considered, either from natural or experimental origin. Previous data were obtained with electric fields applied to individual crayfish (Fossat et al., 2014), but we have verified that this protocol is also suitable for analyzing the consequences of social interactions (Fossat et al., to be published). Three groups of animals should be considered: unstressed or control animals, stressed animals (including stressed animals injected with saline) and stressed animals injected with the anxiolytic drug CDZ.
  2. Before experiment, the pump allowing water oxygenation is switched off and the white LED are switched on, while room light is switched off. The white LED intensity is adjusted to generate approximately a 50-lux intensity in the light arms and a 10-lux intensity in the dark arms.
  3. Each crayfish is carefully fished with a fishnet and placed in an opaque tank placed in the middle of the plus maze (see Figure 4).
  4. The video camera is switched on. A drawing board is filmed during a few seconds to record experiment data (date, reference number of animal).
  5. After at least 1 min, crayfish is released by carefully removing the opaque tank (pierced with small holes to prevent water backwash). Crayfish is allowed to freely visit the entire maze for a period of 10 min.
  6. After 10 min, video camera is switched off. Crayfish is carefully removed from the maze and replaced in its aquarium.
  7. The pump is activated to agitate the freshwater of the maze in all arms for 30 sec. Another crayfish can be tested.
  8. At the end of the experiment, the freshwater is removed from the maze, which is refilled with new freshwater and the pump is activated for oxygenation.
  9. Movies (see Videos 1 to 3) are then analyzed with Ethovision or other tracking software. It is recommended to particularly measure the total distance moved, the time spent in each arm, the number of entries, the latency for the first entry, the mean duration in each arm.
  10. Crayfish often stops moving before entering light arms and then enter or retreat. It is thus possible to estimate a retreat ratio calculated as the number of retreat divided by the total number of attempts.


    Figure 4. An opaque chamber containing the crayfish is placed in the central zone of the plus maze

Representative data



Figure 5. Typical routes of unstressed, stressed and stressed crayfish injected with CDZ. In unstressed, crayfish explore the whole maze (left panel). After a stressful situation, crayfish almost explores the dark arms (center panel). Stressed crayfish injected with CDZ, moved again towards the four arms.


Figure 6. Stress induces a significant light aversion as shown by the decrease of time spent in light arms, the decrease of the mean number of entries in light, the increase of the latency to first entry in light arms and the increase of the retreat ratio (white histograms). Injection of an anxiolytic molecule (i.e., CDZ) significantly abolishes the aversion for light (soft grey histrograms). *** p<0,001, ** p<0,01, one way ANOVA.

Also see movies below:

Video 1. Showing the displacements of a stressed crayfish in the Dark/Light plus maze

Video 2. Showing the displacements of an unstressed crayfish in the Dark/Light plus maze

Video 3. Showing the displacements of a stressed crayfish injected with CDZ in the Dark/Light plus maze

Notes

  1. Be sure that freshwater is well oxygenated and use the pump whenever possible. Stop the pump during experiment.
  2. Between two maze measurements, be sure to correctly agitate water.
  3. When manipulating crayfish, be careful to limit stress. For example, do not test immediately animal that generates repetitive tail flips when fished.
  4. To avoid interference with daily rhythm of the crayfish, perform tests at the same time slot for all the experiments. Behavior can be biased if tested at different time slots.
  5. When removing the opaque chamber, be careful because crayfish can escape by tail flipping. At this point, you can reposition crayfish at the center of the maze and make a mark to note what happened or rather remove the animal and place another one in the maze.
  6. In order to avoid habituation, it seems preferable to test each animal only once. This means that series of control individuals are to be compared to series stressed animals, rather than comparing the behavior of each individual before and after stress.

Acknowledgments

This work was supported by CNRS and “Conseil Régional d’Aquitaine” (2010301037). This protocol is adapted from Fossat et al. (2014).

References

  1. Fossat, P., Bacque-Cazenave, J., De Deurwaerdere, P., Delbecque, J. P. and Cattaert, D. (2014). Comparative behavior. Anxiety-like behavior in crayfish is controlled by serotonin. Science 344(6189): 1293-1297.
  2. 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.
  3. Pellow, S., Chopin, P., File, S. E. and Briley, M. (1985). Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14(3): 149-167.

简介

小龙虾是杂食性的淡水节肢动物,自然地在白天和晚上探索他们的环境,但也经常隐藏在避难所或在危险的洞中。它们可能遭受各种压力,包括捕食,社会互动或环境参数(温度,水质,氧气,等)的变化。最近已经证明,作为压力的结果,小龙虾能够通过限制运动到保护区域来适应其探索行为,反应类似于在啮齿动物中观察到的焦虑样行为(ALB)。为了揭示水生物种的这种行为,我们设计了一个加号形状的亚水生迷宫分为两个保护黑暗的手臂和两个更厌恶的发光武器。这种模式的目的是将小龙虾放置在其对于新颖环境的先天好奇与其对光的厌恶之间的矛盾情境中(Leo,2014; Pellow等人,1985)。未受影响的小龙虾通常探索整个迷宫,包括发光的武器。相比之下,强调的小龙虾优先保持在黑暗的臂中(Fossat等人,2014)。强调小龙虾注射抗焦虑药(利眠宁-CDZ),表现为无压力的动物。与轻武器有关的几个参数可以通过商业软件从视频记录中容易地测量。该方案可以适合于分析任何紧张情况对小龙虾以及许多其他水生物种的ALB的影响。

材料和试剂

  1. 动物
    1. 该研究已在成年雄性小龙虾,Procambarus上进行                          clarkii ,在波尔多(法国)附近在"Réservenaturelle de                      布鲁日"并且在具有12:12暗光循环的动物房子中储存。                      小龙虾随意饲喂颗粒(Novo Prawn)。 使用小龙虾                      对于该研究,为8.7±0.8cm和22.1±1.1g。 我们一般使用                      男性小龙虾在我们的实验(区分他们的第一腹部                      附件),但这个协议也适用于实验                      女性
    2. 在任何实验之前,小龙虾被隔离 丰富的水族箱(75厘米x 40厘米x 50厘米),充满回收                      含氧水和富含鹅卵石和塑料管(后面或                      里面可以隐藏)。 将动物隔离至少3周                      在任何实验之前,为了擦除过去的生活史和                      避免社交互动。
    3. 水必须不含氯                      (搅拌新鲜自来水24小时一般足以去除                      氯)。 每个会话后更换水或使用常规                      水族箱过滤系统。
    4. 鱼网用于操作小龙虾。

  2. 试剂
    1. 林格:小龙虾注射了一个经典的无脊椎动物铃声                      含有195 NaCl,5KCl,13CaCl 2,2MgCl 2和3 HEPES                      (Sigma-Aldrich),pH为7.65
    2. 盐酸利眠宁盐酸盐(CDZ)(Sigma-Aldrich)(溶解于小龙虾环中,并以15μg/g的剂量使用)

设备

  1. 黑暗/光(D/L)加迷宫
    1. 定制装置(65×65cm,参见图1)适应于尺寸                     的成年小龙虾。可能需要更小或更大的尺寸                     小龙虾或其他物种
    2. 照明臂由透明有机玻璃和不透明有机玻璃的暗臂制成(图1)。
    3. 迷宫充满ca。 7 cm氧合淡水。它是                     放置在较大的圆形罐的中心(图2)。圆罐                     (直径1.5m)也填充7cm淡水以平衡                     水压在每个迷宫墙上,并允许良好的氧合和                     回收水(图2)。


      图1.黑暗/光加迷宫。                     迷宫的总长度为65cm×65cm。每个手臂长25厘米,长15厘米 厘米宽。中间区域为15cm×15cm。光是由生成的                     四个源包括两个LED。每个光源都放在                     不透明有机玻璃的外表面。


      图2.黑暗/光加                         迷宫置于较大的罐(直径1.5米)中。 右:房间灯                     打开。左:室内灯在实验条件下关闭。

    4. 光臂的照明由一系列LED执行                     (白光;电压:3.5V,光强度:880〜2530mcd)                     设置在加号迷宫外面,在黑暗的武器的外侧                     最外端(见图3)。


      图3.位于暗臂末端的两个LED照亮一个灯臂。迷宫墙的高度为20厘米。

  2. 其他设备
    1. 摄像机必须放置在迷宫上方。 我们使用handycam(索尼                      inc,型号:HDR-XR155),但可以使用任何商用相机
    2. 在实验阶段之间将泵(NewJet)放置在大罐上以搅拌和氧化水
    3. 用照度计(Chauvin Arnoux)评价光强度。

软件

  1. 我们使用Ethovision v8(Noldus),但可以使用任何跟踪软件。 测量参数列于表1中
    表1.可用于分析D/L加迷宫中焦虑样行为的参数列表
    变量名称(和图2中使用的缩写)
    定义
    测量
    范围
    移动的总距离(距离)
    距离在整个舞台上移动。
    厘米
    ≥0
    %区域中的时间(%T亮,暗)
    在一个区域(黑暗或轻微)/(除以总时间)中花费的时间* 100

    0-100
    延迟到第一次亮起(Lat.Light)
    从开始时间到在轻武器中第一次进入的时间
    第二
    0-600
    在暗处延迟到第一天(Lat.Dark)
    从开始时间到在黑暗的树枝中第一次进入的时间
    第二
    0-600
    轻武器进入平均数(Nb。黑暗)
    从中区到轻武器的进入次数
    NA
    ≥0
    黑暗臂中平均进入数量(Nb。ent。Light)
    从中间区到暗臂的进入次数
    NA
    ≥0
    光臂中每次访问的平均持续时间
    轻武器时间/平均进入次数
    第二
    ≥0
    暗室中每次访问的平均持续时间
    黑暗时间/平均进入次数
    第二
    ≥0
    撤退率(RR)
    撤退次数/总尝试次数
    NA
    0-1

程序

  1. 因为我们的迷宫已被开发来测量压力的后果,小龙虾必须提交一些紧张的情况,然后在黑暗/光加迷宫进行测试。任何压力来源都可以考虑,从天然或实验来源。先前的数据是使用电场应用于单个小龙虾获得的(Fossat等人,2014),但是我们已经验证了该方案也适合于分析社会交互的后果(Fossat等人al。,以便发布)。应考虑三组动物:无应激或对照动物,应激动物(包括注射盐水的应激动物)和注射抗焦虑药物CDZ的应激动物。
  2. 在实验之前,允许水氧合的泵被关闭,并且白色LED被打开,而室内灯被关闭。白光LED强度被调节以在光臂中产生大约50-lux的强度,在暗臂中产生10-lux的强度。
  3. 每个小龙虾用鱼网仔细地捞起并放置在放置在加号迷宫中间的不透明罐中(参见图4)。
  4. 摄像机已打开。在几秒钟内拍摄绘图板以记录实验数据(日期,动物的参考数量)。
  5. 在至少1分钟后,小龙虾通过小心地移除不透明的水槽(穿透小孔以防止水回洗)释放。小龙虾允许自由访问整个迷宫10分钟。
  6. 10分钟后,摄像机关闭。小龙虾被小心地从迷宫中移除,并在其水族馆中更换。
  7. 泵被激活以搅拌所有臂中的迷宫的淡水30秒。另一只小龙虾可以测试。
  8. 在实验结束时,从迷宫中除去淡水,用新的淡水重新填充,并且泵被激活用于氧合。
  9. 然后使用Ethovision或其他跟踪软件分析电影(参见视频1至3)。建议特别测量移动的总距离,每个手臂花费的时间,条目数,第一个条目的延迟时间,每个手臂的平均持续时间。
  10. 小龙虾在进入轻武器之前经常停止移动,然后进入或撤退。因此,可以估计作为撤退次数除以总尝试次数计算的撤退比率。


    图4.包含小龙虾的不透明腔室位于加号迷宫的中心区域

代表数据



图5.注射CDZ的未受胁迫,压力和压力的小龙虾的典型路线。在无压力下,小龙虾探索整个迷宫(左图)。在紧张的情况下,小龙虾几乎探索了黑暗的武器(中图)。强调小龙虾注射CDZ,再次移向四臂。


图6.应力诱导显着的光厌恶,如通过在光臂中花费的时间的减少,光入射的平均数目的减少,光臂中第一次进入的潜伏期的增加和光臂中的第一次进入的增加(白色直方图)。注射抗焦虑分子(,CDZ)显着消除了对光的厌恶(软灰色组织图)。 *** p <0,001,** <0,01,单因素方差分析。
另见下面的电影:

视频1.显示黑暗/光加迷宫中压力小龙虾的位移
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获取Adobe Flash Player

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<! - <![endif] - >
视频2.显示黑暗/光加迷宫中未受胁迫的小龙虾的位移
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获取Adobe Flash Player

<! - [if!IE]> - >
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视频3.显示在黑暗/光加迷宫中注射CDZ的受压小龙虾的位移
                                                                                                                                              <! - [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

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<! - <![endif] - >

笔记

  1. 确保淡水充分氧化,并尽可能使用泵。 实验期间停止泵。
  2. 在两次迷宫测量之间,一定要正确搅拌水。
  3. 操作小龙虾时,小心限制压力。 例如,不要立即测试当捕捞时产生重复尾巴翻动的动物
  4. 为了避免干扰小龙虾的每日节律,在所有实验的同一时间槽进行测试。 如果在不同时间段进行测试,行为可能会出现偏差。
  5. 当去除不透明室时,小心,因为小龙虾可以通过尾部翻转逃脱。 在这一点上,你可以将小龙虾重新定位在迷宫的中心,并做一个标记,注意发生了什么,或者更确切地说,去除动物,并将另一个放在迷宫中。
  6. 为了避免习惯,似乎更好的是测试每只动物只有一次。 这意味着一系列控制个体将与系列压力动物进行比较,而不是比较每个个体在压力之前和之后的行为。

致谢

这项工作得到国家可再生能源研究中心和"阿基坦大学理事会"(2010301037)的支持。该协议改编自Fossat等人(2014)。

参考文献

  1. Fossat,P.,Bacque-Cazenave,J.,De Deurwaerdere,P.,Delbecque,J.P。和Cattaert,D。 比较行为。小龙虾的焦虑样行为由5-羟色胺控制。 344(6189):1293-1297。
  2. Leo,L.M。和Pamplona,F.A。(2014)。 Elevated plus maze test to assess anxiety-like behavior in the mouse。 Bio-protocol 4(16):e1211。
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引用:Fossat, P., Bacqué-Cazenave, J., Delbecque, J. and Cattaert, D. (2015). Measuring Anxiety-like Behavior in Crayfish by Using a Sub Aquatic Dark-light Plus Maze. Bio-protocol 5(3): e1396. DOI: 10.21769/BioProtoc.1396.
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