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Delayed-matching-to-place Task in a Dry Maze to Measure Spatial Working Memory in Mice
采用日间迷宫中延迟位置匹配任务来衡量小鼠的空间工作记忆   

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Abstract

The delayed-matching-to-place (DMP) dry maze test is a variant of DMP water maze (Steele and Morris, 1999; Faizi et al., 2012) which measures spatial working/episodic-like learning and memory that depends on both hippocampal and cortical functions (Wang and Morris, 2010; Euston et al., 2012). Using this test we can detect normal aging related spatial working memory decline, as well as trauma induced working memory deficits. Furthermore, we recently reported that fractionated whole brain irradiation does not affect working memory in mice (Feng et al., 2016). Here we describe the experimental setup and procedures of this behavioral test.

Keywords: DMP dry maze(DMP干迷宫), Working memory(工作记忆), Behavior(行为), Mouse(小鼠)

Background

The reference-memory water maze (RMW) was originally used to measure spatial reference memory in rats. In this task animals are trained to find a hidden platform in a fixed location under opaque water by using distal clues outside of the water maze (Morris, 1981). Over the years it has evolved into various tasks that allow probe trials, over training, reverse learning and an on-demand platform (Morris et al., 1982; Morris et al., 1990; Spooner et al., 1994; Lipp and Wolfer, 1998). Later the Morris lab developed a delayed matching-to-place (DMP) water maze that requires frequently updated, ‘delayed’ memory of escape locations in an unchanging environment (Steele and Morris, 1999). These variations of Morris water maze (MWM) are widely used in the neuroscience field to study spatial cognitive functions that involve different brain regions in both rats and mice (Vorhees and Williams, 2006). The main concern for these tests is that forced swimming might induce stress for animals (Vorhees and Williams, 2014). To exclude this limitation Faizi et al. (2012) designed a dry maze based on the principles of the DMP water maze. The DMP dry maze is believed to measure the same working/episodic-like memory as the DMP water maze with a less intense test paradigm for both the experimenter and test subjects.

Materials and Reagents

  1. Paper towels (Renown, catalog number: REN06116-WB )
  2. Red paper towels (KCWW, Kimberly-Clark, catalog number: 05930 )
  3. C57BL/6J male adult mice (3-18 months of age)
    Note: C57BL/6J male adult mice (3-18 months of age) housed in a room with reversed light cycle for at least two weeks prior to test. Behavior experiment is conducted during the dark cycle (7 AM-7 PM, see Note 1).
  4. 70% ethanol (v/v in ddH2O) in a spray bottle

Equipment

  1. A well-lit (1,200 lux) behavior room isolated from noises and a close-by holding room (Figure 1A)
  2. White shower curtains (Figures 1B and 1C)
  3. Two large visual clues (Figures 1B and 1C)
  4. A Polystyrene circular DMP dry maze platform (Diameter = 122 cm, thickness = 1.2 cm) with 40 escape holes (D = 5 cm)
    Note: 16 holes on the outer ring, 16 on the middle ring and 8 holes on the inner ring with distance of 50, 35 and 20 cm to the center of platform, respectively (Figure 1B). ABS tubes (Inner diameter = 52 mm, outer diameter = 60 mm) are attached to each escape hole which allows easy attach and detach of the escape tube (Figure 1D). It is important to use black or dark colored escape tube so mice would prefer to enter. In addition, dark color minimizes the chance of leaving visual clues around escape holes over time.
  5. A 3” ABS plug (NIBCO, catalog number: 5818 )
  6. Escape tube assembled using black ABS pipes (2”, NIBCO, catalog numbers: C5806-2 and C5807-V Figure 1E) with a removable plug (to be attached to escape holes) at the other end (NIBCO, catalog number: 5818 , Figure 1F). When connected to the escape hole, the resulting depth from the top pf maze to the floor of escape tube is about 8 cm
  7. A metal stand to support the platform to 90 cm above the floor (Figure 1 D)
  8. A small non-transparent transfer box
    Note: We use a pipette tip box without the lid (Figure 1I).
  9. Fish net with extended handle or a similar item (Figure 1I)
  10. Timer
  11. Speakers capable of playing at 85 dB or louder (Figure 1B)
  12. Noise-cancelling headphones or similar items (Figure 1I)
  13. Overhead camera (GigE, catalog number: XCFS-BC6o# )
  14. A computer (with Windows 7 64bit Professional) connected to the camera


    Figure 1. Platform and room setup. A. Sketch of the behavior room and holding room layout; B. A picture showing the details of platform layout and visual clues on two sides of the platform; C. A picture of visual clues on other two sides; D. A picture showing the bottom of platform; E-H. Pictures to show the escape tube, an escape hole and how they are connected; I. Other items needed for the task.

Software

  1. Recording and tracking software (Noldus, Ethovision XT v 11.5.1026)
  2. Audio file of a recorded tone at 960 Hz with > 90 sec length (audio file 1)

Procedure

  1. Software setup (Ethovision v 11.5.1026)
    Note: Instructions launch automatically when a new experiment is created. Here is a brief description of DMP dry maze experiment setup.
    1. In the ‘Arena Settings’ take a snapshot from the overhead camera. Make sure the arena is at the center of camera view, zoom in to maximize the size of the DMP dry maze platform in visible window on the computer screen (Figure 2).


      Figure 2. Arena, escape holes and calibration setup. The Arena is set to an area slightly bigger than the DMP dry maze platform. The platform diameter (122 cm) is used to calibrate the bit size of recorder videos. Escape holes were changed daily but kept the same for trials conducted on the same day.

    2. Use the ‘Draw Scale to Calibrate’ tool in ‘Arena Settings’ to draw a line (scale) to cover the diameter of platform. The scale size is set to 122 cm. Use the draw tools to draw an arena to cover the entire DMP dry maze platform. Draw a circle to cover the escape hole. Circle size can be adjusted by double-click and input the width and height manually. Circle size is set to 7.0 x 7.0 cm.
      Note: It is important to set the arena bigger than the platform. Setting the arena to exactly the size of platform results in loss of tracking if mouse moves along the edge.
    3. In the ‘Trial Control Settings’ define conditions for track start, stop and end (Figure 3). Automatic tracking should start 5 sec after the software detects a mouse. Trial ends after 90 sec of tracking or immediately after mouse enters the escape hole (defined as both the nose-point and center-point of a mouse are in the escape hole).
    4. Switch to the ‘Detection Settings’ page; connect the escape tube to the designated escape hole, put a control mouse of the same color and size as test subjects on the platform. Adjust contrast settings to ensure good tracking.


      Figure 3. Trial control and experimental timeline. A. Recording begins after the experimenter clicks Start before each planed trial. Track start condition is set to ‘cumulative duration over 5 sec when center-point is in arena’. Track will stop after one of the following conditions is met, a) Escaping, defined as mouse ‘center-point is in Escape Target Hole Day N’, b) Time limit, defined as ‘After a delay of 90 sec’. The recording ends immediately after tracking is ended. B. Schematic chart to demonstrate the experimental timeline.

  2. DMP task
    1. Preparation
      1. One week before the task mice are tail marked and briefly handled by the experimenter every day for 5 days (see Note 2).
      2. On each of the test days, mice are placed in the holding room (across the hallway) in their home cage kept in dark (see Note 3) at least one hour prior to the first trial.
      3. Each mouse was habituated for 2 min in the escape tube before beginning the first trial on the first test day (see Note 2).
    2. Test Day 1
      1. Attach the escape tube to the designated escape hole.
      2. Put the mouse in the small transfer box and cover with a red paper towel. Bring it to the test room. Put the mouse on the platform at the center and keep it covered by the transfer box.
      3. After a delay of about 30 sec, start recording in the Ethovision software, turn on the tone noise (played at 85 dB, see Note 4) and immediately remove the transfer box to expose mouse in the environment. The experimenter then moves to the recording side of the room and monitors the tracking from computer screen (see Figure 3 for the trial control settings).
      4. Wait until the mouse completely enters the escape tube. Stop the tone noise and cover the escape hole with the 3” ABS plug (see Notes 5 and 6). After a 10 sec delay detach the tube, cover it with a red paper towel and put the mouse back to its home cage. Set the timer to count down from 10 min.
      5. Clean the escape tube and the platform with 70% ethanol and wipe with paper towels. Start the next trial when the timer counts down to zero.

        Note: To be more efficient we carried out the experiment in a paired way (Figure 3B).

    3. Test Days 2-4 (see Note 7)
      Change the escape hole to a new location (Figure 2) on each of the following test days. Repeat trials using the same procedures as Test Day 1.
      Note: Escape hole location is kept the same for all the mice on each day. A total of four different locations are used (see Figure 2).

Data analysis

  1. Set the analysis profile in Ethovision software, and measure the following parameters: a) velocity of the center-point; b) when nose-point and center-point are in the escape hole (see escape holes in Figure 2); c) Trial control state to cover the period during which trials are active.
  2. Extract velocity data of the center-point within each experimental group. Low velocity during trials indicates that the animal either has difficulty in moving or lack of motivation to enter the escape hole. Therefore, all data from the outlier should be excluded from the analysis. We rarely find outlier in the Closed Head Injury (CHI) (Lloyed et al., 2008) and Whole-brain irradiation models (Feng et al., 2016).
  3. Pull out the data on ‘Latency to Escape’ defined as the time between trial start and when nose-point and center-point are in the escape hole. Figure 4 shows an example of working memory impairment in animals received CHI.
    Note: Alternatively, ‘primary error number can be used’. This method measures the number of visit to non-escape holes before the mouse finds the escape hole. Results should have similar trends as those of latency to escape.


    Figure 4. Working memory is impaired in old mice compared to young (A) and in young mice after CHI (B). A. Young (3 months) and old (18 months) mice have similar performance on day 1 and day 2 but old mice show significantly less trial by trial improvement on day 3 and day 4. B. Working memory deficit were observed in young mice 2 weeks after CHI (mice were 14 weeks old at the time of behavior test). Sham animals show progressive improvement while CHI animals are unable to learn over time (*P < 0.05, **P < 0.01, ***P < 0.001. Statistics: Data were analyzed using two-way ANOVA of repeated measures; P-values are calculated by Tukeys multiple comparisons test).

  4. Check the tracking using Ethovision ‘Analysis’-‘Results’-‘Track Visulization’, after clicking the ‘play’ button the software will automatically generate a travelling path for each trial.
  5. Animals that learned and remembered the escape hole usually have progressive ‘learning’ showed as shorter tracks trial by trial on day 2-4 (Figure 5).


    Figure 5. Comparison of tracks of sham and CHI animals during DMP dry maze task on Day 3. A sham mouse found the escape hole location on the first trial on day 3 (A), remembered it in the following trials, and entered quickly in following trials (B-D). A CHI mouse did not find the escape hole on the first two trials (E and F), accidentally found it on trial 3 (G) but failed to locate and enter on trial 4 (H). Escape holes are marked in red circles.

Notes

  1. We choose to perform this task during the dark cycle because mice are supposed to be more active and more sensitive to strong light exposure.
  2. Handling mice before experiment eliminates anxiety caused by the experimenter. Our experimental procedure requires a short travel between two separate rooms. Mice are carried to the maze in a small box covered by a piece of red paper towel before a trial. Upon completion of each trial mice are carried back to their cage in the escape tube. Therefore, during the handling the experimenter would pick up the mouse, put it in the box, cover with red paper towel, walk around and put it back to its home cage. In addition, each mouse is habituated in the escape tube for about 2 min before the beginning of the first trial of test day 1. The experimenter picks up a mouse, lets it climb into the escape tube, covers it with a piece of red paper towel, hold it still for about 10 sec, walks around and puts the mouse back into its home cage. In principle, the handling is designed to allow the mice to get familiar with the experimenter and the trial procedures.
  3. Protecting the holding cages from light is essential as strong light provides aversive environment and motivation for mice to enter the escape tube. Pre-exposure to light can weaken this response. Similarly, tone noise during the trials must not be heard in the holding room. While shorter habituation time (e.g., 20 or 30 min) is also used in behavior assays, we have always allowed one hour to further minimize stress. The longer habituation does not affect test results in our hands.
  4. Prolonged exposure to loud noise is considered hazardous to humans. Noise Cancelling headphones or ear plugs are necessary.
  5. On the first day trial one some mice might not find the escape hole/tube in 90 sec. If this happens the experimenter should use the fishing net to gently guide the mouse to the escape hole and let it enter the escape tube by tabbing around the mouse with the tone noise on.
  6. A mouse might show hesitancy to enter the escape tube during trials, shown as poking its head into the tube with its palms at the edge of the escape hole, and part of the body leaning towards in the escape tube without fully entering. This might happen on test days 3 and 4 (about 3% of total trials), which could be caused by hind limb problems (occasionally seen in CHI models), anxiety or lack of motivation (animals get used to the test environment on latter test days). Software might continue tracking when the mouse moves away. However, the trial should be re-analyzed to correct the latency to escape. See an example in Video1. The test mouse finds the escape hole without entering; it leaves and comes back again. The trial is manually ended when the head fully enters the escape hole for the first time. Ethovision settings can be adjusted to account for this possibility. This can also occur if there are mobility problems with the animal and entry into the tunnel is painful or difficult. In this case, the animal should be excluded for subsequent testing. If this is found to happen more regularly it may help to handle the animals longer, habituate them longer to the escape tube.

    Video 1. An example of the test mouse finds escape hole without entering

Acknowledgments

This protocol was used in our previous study to measure working memory performance in mice (Feng et al., 2016). This work was supported by NIH grants R01 CA133216 and R01 CA213441 to SR.

References

  1. Euston, D. R., Gruber, A. J. and McNaughton, B. L. (2012). The role of medial prefrontal cortex in memory and decision making. Neuron 76(6): 1057-1070.
  2. Faizi, M., Bader, P. L., Saw, N., Nguyen, T. V., Beraki, S., Wyss-Coray, T., Longo, F. M. and Shamloo, M. (2012). Thy1-hAPPLond/Swe+ mouse model of Alzheimer's disease displays broad behavioral deficits in sensorimotor, cognitive and social function. Brain Behav 2(2):142-154.
  3. Feng, X., Jopson, T. D., Paladini, M. S., Liu, S., West, B. L., Gupta, N. and Rosi, S. (2016). Colony-stimulating factor 1 receptor blockade prevents fractionated whole-brain irradiation-induced memory deficits. J Neuroinflammation 13(1): 215.
  4. Lipp, H. P. and Wolfer, D. P. (1998). Genetically modified mice and cognition. Curr Opin Neurobiol 8(2): 272-280.
  5. Lloyd, E., Somera-Molina, K., Van Eldik, L. J., Watterson, D. M. and Wainwright, M. S. (2008). Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury. J Neuroinflammation 5: 28.
  6. Morris, R. G., Garrud, P., Rawlins, J. N. and O'Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature 297(5868): 681-683.
  7. Morris, R. G. M. (1981). Spatial localization does not require the presence of local cues. Learn Motiv 12:239-260.
  8. Morris, R. G., Schenk, F., Tweedie, F. and Jarrard, L. E. (1990). Ibotenate lesions of hippocampus and/or subiculum: dissociating components of allocentric spatial learning. Eur J Neurosci 2(12): 1016-1028.
  9. Spooner, R. I., Thomson, A., Hall, J., Morris, R. G. and Salter, S. H. (1994). The Atlantis platform: a new design and further developments of Buresova's on-demand platform for the water maze. Learn Mem 1(3): 203-211.
  10. Steele, R. J. and Morris, R. G. (1999). Delay-dependent impairment of a matching-to-place task with chronic and intrahippocampal infusion of the NMDA-antagonist D-AP5. Hippocampus 9(2): 118-136.
  11. Vorhees, C. V. and Williams, M. T. (2006). Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 1(2): 848-858.
  12. Vorhees, C. V. and Williams, M. T. (2014). Assessing spatial learning and memory in rodents. ILAR J 55(2): 310-332.
  13. Wang, S. H. and Morris, R. G. (2010). Hippocampal-neocortical interactions in memory formation, consolidation, and reconsolidation. Annu Rev Psychol 61: 49-79, C41-44.

简介

延迟匹配(DMP)干燥迷宫测试是DMP水迷宫(Steele和Morris,1999; Faizi等人,2012)的变体,其测量空间工作/情景样 依赖于海马和皮质功能的学习和记忆(Wang和Morris,2010; Euston等人,2012)。 使用这个测试,我们可以检测到正常老化相关的空间工作记忆衰退,以及创伤引起的工作记忆缺陷。 此外,我们最近报道,分离的全脑照射不影响小鼠的工作记忆(冯等人,2016年)。 在这里我们描述这个行为测试的实验设置和程序。
【背景】参考记忆水迷宫(RMW)最初用于测量大鼠的空间参考记忆。在这个任务中,通过使用水迷宫之外的远端线索,动物被训练成在不透明水域的固定位置找到隐藏的平台(Morris,1981)。多年来,它已经演变成允许探针试验,过度训练,反向学习和按需平台的各种任务(Morris等人,1982; Morris等人,,1990; Spooner等人,1994; Lipp和Wolfer,1998)。后来,莫里斯实验室开发了一个延迟的匹配地点(DMP)水迷宫,需要在不断变化的环境中经常更新,延迟记录逃生地点(Steele和Morris,1999)。 Morris水迷宫(MWM)的这些变化被广泛用于神经科学领域,以研究涉及大鼠和小鼠中不同脑区的空间认知功能(Vorhees和Williams,2006)。这些测试的主要担忧是强迫游泳可能会诱发动物压力(Vorhees and Williams,2014)。为了排除这一限制,Faizi等人(2012)基于DMP水迷宫的原理设计了一个干迷宫。 DMP干迷宫被认为是测量与DMP水迷宫相同的工作/情景状态的记忆,对于实验者和测试对象,测试范例不太强烈。

关键字:DMP干迷宫, 工作记忆, 行为, 小鼠

材料和试剂

  1. 纸巾(Renown,目录号:REN06116-WB)
  2. 红纸巾(KCWW,Kimberly-Clark,目录号:05930)
  3. C57BL / 6J雄性成年小鼠(3-18个月龄)
    注意:C57BL / 6J雄性成年小鼠(3-18个月龄)在测试前至少两周放置在具有反向光循环的房间中。行为实验在黑暗周期(7 AM-7 PM,见注1)进行。
  4. 70%乙醇(v / v,ddH 2 O)在喷雾瓶中

设备

  1. 一个光线充足的(1,200勒克斯)行为室隔离了噪音和一个靠近的房间(图1A)
  2. 白色浴帘(图1B和1C)
  3. 两个大视觉线索(图1B和1C)
  4. 具有40个逃生孔(D = 5cm)的聚苯乙烯圆形DMP干迷宫平台(直径= 122cm,厚度= 1.2cm)
    注意:外圈16个孔,中间环16个,内环8个孔,距离平台中心距离为50,35和20厘米(图1B)。 ABS管(内径= 52mm,外径= 60mm)安装在每个逃生孔上,这样可以方便地安装和拆卸逃生管(图1D)。使用黑色或深色的逃生管是重要的,所以老鼠宁愿进入。此外,深色可以最大限度地减少随着时间的推移,在逃生孔周围留下视觉线索的机会。
  5. 3“ABS插头(NIBCO,目录号:5818)
  6. 使用黑色ABS管(2“,NIBCO,目录号:C5806-2和C5807-V图1E)组装的逃生管,另一端带有可拆卸插头(要连接到逃生孔)(NIBCO,目录号:5818,图1F)。当连接到逃生孔时,从顶部pf迷宫到逃生管的地板的深度约为8cm
  7. 一个金属支架支撑平台在地板上方90厘米(图1 D)
  8. 一个小的不透明的转箱
    注意:我们使用没有盖子的移液器吸头盒(图1I)。
  9. 带有扩展手柄或类似物品的鱼网(图1I)
  10. 计时器
  11. 扬声器能够以85dB或更大的音量播放(图1B)
  12. 降噪耳机或类似物品(图1I)
  13. 架空摄像机(GigE,目录号:XCFS-BC6o#)
  14. 连接到相机的计算机(Windows 7 64位专业版)


    图1.平台和房间设置。 A.行为房间的示意图和房间布局; B.展示平台两侧平台布局和视觉线索细节的图片; C.其他双方视觉线索的图片; D.展示平台底部的图片; E-H。图片显示逃生管,逃生孔及其连接方式; I.任务所需的其他项目。

软件

  1. 录音和跟踪软件(Noldus,Ethovision XT v 11.5.1026)
  2. 具有960Hz的记录音的音频文件具有&gt;长度为90秒(音频文件1

程序

  1. 软件设置(Ethovision v 11.5.1026)
    注意:创建新实验时,会自动启动指令。这是DMP干迷宫实验设置的简要说明
    1. 在“竞技场设置”中,从高架摄像头拍摄快照。确保竞技场处于相机视图的中心,放大以最大化计算机屏幕上可见窗口中DMP干迷宫平台的大小(图2)。


      图2.竞技场,逃生孔和校准设置。 竞技场设置为比DMP干迷宫平台稍大的区域。平台直径(122厘米)用于校准录像机视频的位大小。逃生孔每日更换,但在同一天进行的试验保持不变。

    2. 使用“竞技场设置”中的“绘制比例校准”工具绘制线(刻度)以覆盖平台的直径。刻度尺设置为122厘米。使用绘图工具绘制一个竞技场,以覆盖整个DMP干迷宫平台。画一个圆圈来覆盖逃生孔。可以通过双击并手动输入宽度和高度来调整圆圈大小。圆形尺寸设置为7.0 x 7.0厘米。
      注意:设置舞台比平台更重要。如果鼠标沿着边缘移动,将竞技场设置为正确的平台大小导致跟踪失去。
    3. 在“试验控制设置”中定义轨道启动,停止和结束的条件(图3)。自动跟踪应该在软件检测到鼠标5秒后开始。试验结束后90秒跟踪或鼠标进入逃生孔后(定义为鼠标的鼻点和中心点都在逃生孔中)。
    4. 切换到“检测设置”页面;将逃生管连接到指定的逃生孔,将与测试对象相同颜色和大小的控制鼠标放在平台上。调整对比度设置以确保良好的跟踪。


      图3.试用控制和实验时间表。 :一种。实验者在每次计划试用之前点击“开始”开始记录。轨道起始条件设置为“中心点在舞台上的累积持续时间超过5秒”。轨道将在满足以下条件之一后停止:a)转义,定义为鼠标“中心点处于逃脱目标孔日N”,b)时间限制,定义为“延迟90秒”。跟踪结束后立即结束记录。 B.演示实验时间表的示意图。

  2. DMP任务
    1. 制备
      1. 任务前一周,小鼠每天尾部标记并由实验者短暂处理5天(见注2)。
      2. 在每个测试日期,至少在第一次试验前一小时,将小鼠放置在保持在黑暗中的家庭护栏(横过走廊)(见注3)。
      3. 在第一个测试日开始第一次试验前,每只小鼠在逃生管中习惯2分钟(见注2)。
    2. 测试日1
      1. 将逃生管连接到指定的逃生孔。
      2. 将鼠标放在小的传输盒中,并用红色的纸巾盖住。带到测试室。将鼠标放在中心的平台上,并将其保留在传输盒中。
      3. 延迟约30秒后,在Ethovision软件中开始录音,打开音色(以85 dB播放,见附注4),并立即取出传送盒,将鼠标暴露在环境中。实验者然后移动到房间的记录侧,并从计算机屏幕监视跟踪(参见图3的试用控制设置)。
      4. 等到鼠标完全进入逃生管。停止音色,并用3“ABS插头盖住逃生孔(见注5和6)。延迟10秒钟后,将管子取下,用红色的纸巾盖住,然后将鼠标放回家中。设置定时器从10分钟倒数。
      5. 用70%乙醇清洁逃生管和平台,并用纸巾擦拭。当定时器倒数为零时,开始下一个试用。

        注意:为了更有效率,我们以配对的方式进行实验(图3B)。

    3. 测试日2-4(见注7)
      在以下每个测试日期,将逃生孔更改为新位置(图2)。使用与测试日1相同的步骤重复试验。
      注意:每一天,所有小鼠的逃生孔位置保持不变。总共使用四个不同的位置(见图2)。

数据分析

  1. 在Ethovision软件中设置分析概况,并测量以下参数:a)中心点的速度; b)当鼻尖和中心点在逃生孔中时(见图2中的逃生孔); c)审判控制状态,以涵盖试验活动期间。
  2. 提取每个实验组中心点的速度数据。试验中的低速度表明,动物难以移动或缺乏进入逃生孔的动机。因此,来自异常值的所有数据都应该从分析中排除。我们很少发现闭合性头部损伤(CHI)(Lloyed et al。,2008)和全脑照射模型(Feng等人,2016)的异常值。
  3. 拉出“延迟退出”的数据,定义为试用开始之间的时间和鼻尖和中心点在逃生孔中的时间。图4显示了接受CHI的动物工作记忆障碍的一个例子。
    注意:或者,可以使用“主要错误号”。该方法测量在鼠标找到逃生孔之前访问非逃生孔的次数。结果应该具有与逃避潜伏期相似的趋势。


    图4.老年小鼠相对于年轻(A)和CHI(B)后的年轻小鼠中,工作记忆受损。 :一种。年龄(3个月)和老年(18个月)小鼠在第1天和第2天具有相似的表现,但是老鼠在第3天和第4天显示出较少的试验改善。B.在小鼠中观察到工作记忆缺陷2周CHI后(小鼠在行为测试时为14周龄)。假动物显示渐进性改善,而CHI动物随着时间的推移无法学习(* <0.05,** P <0.01,*** < / em>&lt; 0.001统计:使用重复测量的双向方差分析来分析数据;通过Tukeys多重比较测试计算P 值。

  4. 使用Ethovision'Analysis' - 'Results' - 'Track Visulization'检查跟踪,点击“播放”按钮后,软件将自动为每个试验生成一条旅行路线。
  5. 学习和记住逃生洞的动物通常在第2-4天通过试用显示为渐进的“学习”(图5)。


    图5.第3天DMP干燥迷宫任务期间假手术和CHI动物的轨迹比较 假手小鼠在第3(A)天在第一次试验中发现逃生孔位置,记得在以下试验,并在以下试验(BD)中迅速进入。在前两次试验(E和F)中,CHI小鼠没有发现逃生孔,在试验3(G)上意外发现,但未能定位并进入试验4(H)。逃生洞被标记为红色圆圈。

笔记

  1. 我们选择在黑暗循环期间执行这项任务,因为老鼠应该更加活跃,对强烈的光线曝光更敏感
  2. 实验前处理小鼠消除了实验者引起的焦虑。我们的实验程序需要在两个单独的房间之间短暂的行程。在试用前,将小鼠携带到一块红色纸巾上的一个小盒子的迷宫中。在每次试验完成后,将小鼠携带回到逃逸管中的笼中。因此,在处理过程中,实验者将拿起鼠标,将其放在盒子里,用红纸巾盖住,然后回到家中的笼子里。另外,在试验第一天的第一次试验开始之前,每只小鼠在逃生管中习惯约2分钟。实验者拿起一只小鼠,让它爬进逃生管,用一张红纸覆盖毛巾,持续约10秒钟,走动,将鼠标放回家中的笼子里。原则上,处理设计是为了让老鼠熟悉实验者和试用程序。
  3. 保护保持笼子免受光线的影响是至关重要的,因为强光可以为老鼠进入逃生管提供厌恶的环境和动力。预曝光会削弱这种反应。同样地,在试验期间的音调噪音不能在保持室听到。虽然较短的习惯时间(例如,20或30分钟)也用于行为测定,但我们总是允许一个小时以进一步最小化压力。更长的习惯不会影响我们手中的测试结果。
  4. 长时间暴露于大噪声被认为对人类有害。降噪耳机或耳塞是必要的。
  5. 在第一天试用中,一些老鼠在90秒内可能找不到逃生孔/管。如果发生这种情况,实验者应该使用钓鱼网轻轻地将鼠标引导到逃生孔,并通过在铃声上贴上鼠标来进入逃生管。
  6. 在试验期间,鼠标可能会犹豫进入逃生管,显示为将其头部浸入管中,其手掌位于逃生孔的边缘,部分身体倾斜到逃生管中,而不会完全进入。这可能发生在测试第3天和第4天(总试验的约3%),这可能是由后肢问题(CHI模型偶尔见到),焦虑或缺乏动机(动物习惯于后期测试的测试环境)引起的天)。当鼠标移开时,软件可能会继续跟踪。然而,应该重新分析试验以纠正逃跑的延迟。参见Video1中的一个例子。测试鼠标找不到进入的逃生孔;它离开又回来了。当头部首次完全进入逃生孔时,手动手动结束。可以调整Ethovision设置以考虑到这种可能性。如果动物存在流动性问题,进入隧道也是痛苦或困难的,也可能发生。在这种情况下,应排除动物进行后续检测。如果发现这种情况发生得更加规律,那么可能会有助于更长时间地处理动物,从而使他们更长时间地逃离逃生管。

    Video 1. An example of the test mouse finds escape hole without entering

    To play the video, you need to install a newer version of Adobe Flash Player.

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致谢

我们以前的研究中使用了该方案来测量小鼠的工作记忆性能(Feng等人,2016)。这项工作得到NIH授权R01 CA133216和R01 CA213441对SR的支持。

参考

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引用:Feng, X., Krukowski, K., Jopson, T. and Rosi, S. (2017). Delayed-matching-to-place Task in a Dry Maze to Measure Spatial Working Memory in Mice. Bio-protocol 7(13): e2389. DOI: 10.21769/BioProtoc.2389.
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