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Barnes Maze Procedure for Spatial Learning and Memory in Mice

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The Journal of Neuroscience
Nov 2015



The Barnes maze is a dry-land based rodent behavioral paradigm for assessing spatial learning and memory that was originally developed by its namesake, Carol Barnes. It represents a well-established alternative to the more popular Morris Water maze and offers the advantage of being free from the potentially confounding influence of swimming behavior. Herein, the Barnes maze experimental setup and corresponding procedures for testing and analysis in mice are described in detail.

Keywords: Spatial memory (空间记忆), Mouse (老鼠), Hippocampus (海马), Cognition (认识), Behavior (行为)


The Barnes maze is a dry-land based behavioral test that was originally developed by Carol Barnes to study spatial memory in rats (Barnes, 1979) and later adapted for use in mice (Bach et al., 1995). Conceptually, it is similar to the Morris water maze (MWM) (Morris, 1984), in that it is a hippocampal-dependent task where animals learn the relationship between distal cues in the surrounding environment and a fixed escape location. For mice, the typical Barnes maze setup consists of an elevated circular platform with 40 evenly-spaced holes around the perimeter. An escape tunnel is mounted underneath one hole while the remaining 39 holes are left empty. Both bright light and open spaces are aversive to rodents, thus serve as motivating factors to induce escape behavior. The escape tunnel is maintained at a fixed location for the duration of training, which involves multiple daily trials spread over several days. During the course of training, rodents typically utilize a sequence of three different search strategies (random, serial, spatial) to learn the location of the escape tunnel. Following sufficient acquisition training, the escape tunnel is removed and a probe trial is administered to assess spatial reference memory.

Although the MWM is the dominant model for assessing spatial learning in rodents, the Barnes maze offers several important advantages worth noting. First and foremost, the Barnes maze does not involve swimming and the potential confounding factors associated with it. Swimming is stressful, as detailed in studies documenting that MWM training increases plasma corticosterone levels to a greater extent than that of the Barnes maze (Harrison et al., 2009). In addition, the swim conditions utilized in most MWM protocols elicit reductions in core body temperature that can affect performance (Iivonen et al., 2003). Moreover, rodents often take to floating, which is thought to represent a state of behavioral despair and is considered an index of ‘depressive-like’ behavior in the widely utilized Porsolt forced swim test (Porsolt et al., 1977). Finally, as noted above, the Barnes maze allows clear delineation of the three possible search strategies used by the mouse during performance of each trial.

Materials and Reagents

  1. Tissue paper (Georgia-Pacific Consumer Products, catalog number: 48100 )
  2. 70% ethanol in a spray bottle
  3. C57BL/6J adult male mice (Purchased from Jackson Labs, 3-5 months of age)


  1. Well-lit (~1,000 lux) testing room with a holding room located nearby (Figure 1A)
  2. Barnes maze apparatus (TSE Systems, catalog number: 302050-BM/M ), includes:
    1. Circular PVC platform* (diameter = 122 cm; thickness = 1 cm) containing 40 equally spaced holes (diameter = 5 cm) (Figure 1B)
    2. Gray PVC start chamber* consisting of a base plate and a cover (Figure 1C)
    3. PVC escape tunnel* that can be mounted under any of the 40 escape holes (Figure 1D)
    4. Aluminum support frame* (height = 80 cm) for circular PVC platform (Figure 1E)
  3. Overhead camera (Panasonic, catalog number: WV-BP332 , Figure 1F)
  4. Three distal visual cues (length/width ~30 cm) surrounding the platform (Figure 1G)
  5. Loudspeaker for 90 dB white noise (Sony, catalog number: SS-MB150H )
  6. Windows-based PC computer (Dell, model: OptiPlex 780 ) connected to the camera
  7. Tally counter

    Figure 1. Barnes maze experimental setup. A. Layout of behavioral testing room and adjacent room used for analysis. B-F. Images of the Barnes maze platform (B), start chamber (C), escape tunnel (D), aluminum support frame (E), overhead camera (F), a single visual cue (G).


  1. TSE VideoMot2 video tracking software (TSE Systems)
  2. GraphPad Prism version 5.0 (GraphPad Software)
  3. Microsoft Excel


  1. Software setup
    1. Calibration
      1. Under the ‘Mode’ tab located in the upper left corner of VideoMot2 software program, select ‘Calibration’ (Figure 2).
      2. Hit the button for ‘Calibration’.
      3. Draw a line across the diameter of the Barnes maze platform.
      4. Enter 1,220 mm in the box for ‘Real length’. This corresponds to the actual diameter of the Barnes maze.

        Figure 2. Screenshot of VideoMot2 analysis software

    2. Define experimental region
      1. Under the ‘Mode’ tab in the VideoMot2 program, select ‘Experimental regions’.
      2. Use the draw tools located at the bottom of the display window to make a circle that covers the entire Barnes platform. This defines the region where the mouse will be tracked by the software.
    3. Define informational region
      1. Under the ‘Mode’ tab in the VideoMot2 program, select ‘Informational regions’.
      2. Use the draw tools to make a circle around the hole covering the escape tunnel.
    4. Measurement
      1. Select ‘Measurement’ from the ‘Mode’ tab.
      2. Enter 00:03:00 in the white box to the right of the text ‘Autostop at’. Make sure that the white box to the left of the ‘Autostop at’ text is checked off. This will insure that the video tracking software automatically stops each trial after 3 min.
      3. Click on the ‘Start’ button located in the lower right corner of the display window.
      4. Enter relevant study information (Study number, etc.) when prompted.
      5. Click the spacebar once to start the background measurement and then again to conclude it. During the background measurement, make sure that there is no movement or change in lighting in view of the camera.
      6. Enter trial data (Animal #, Group) when prompted.
      7. Click the spacebar once to begin tracking the mouse. In the video display, a cross will be superimposed on the mouse’s body and will follow its movements in the maze. Click the spacebar again to end the trial.
      8. To save data for each trial, select ‘Save track as’ under the ‘File’ tab located in the upper left corner of the screen.

  2. Barnes maze procedure (Figure 3)
    1. Habituation (Day 1)
      1. Attach the escape tunnel to the platform and add one piece of clean tissue paper for each mouse habituation trial. The surrounding visual cues should be in place. These cues should remain unaltered for the habituation, acquisition, and probe trial phases.
      2. Place mouse in the escape tunnel for 1 min.
      3. Put mouse in the center of the apparatus. Allow it to explore until it enters the escape tunnel or 5 min elapses.
      4. Clean the apparatus and escape tunnel with 70% ethanol
    2. Acquisition training (Days 1-10)
      1. Allow an interval of at least 1 h between habituation and the onset of acquisition training. Attach the escape tunnel to the platform at a location different from that used for the habituation trial. The position of the escape tunnel remains at this fixed location relative to spatial cues in the room for the duration of training. Add one piece of clean tissue paper to the escape tunnel for each mouse acquisition trial.
      2. Training consists of two acquisition trials daily (3 min limit per trial; intertrial interval ~1 h) with the starting location varied pseudorandomly among the four quadrants.
      3. At the start of each trial, the mouse is placed in a gray PVC start chamber located in the center of one of the four quadrants. After a 15 sec period, the start chamber is lifted (Figure 3B), and the mouse is allowed to explore the maze. During each trial, loud white noise (90 dB) is played through a loudspeaker to induce escape behavior. The trial concludes when the mouse enters the escape tunnel (Figure 3D) or 3 min elapses. If a mouse fails to find the escape tunnel within the 3 min period, it is placed in the tunnel by the researcher and allowed to stay there for 15 sec prior to removal.

        Figure 3. Barnes maze procedure. A. Experimental timeline; B-D. Images of a mouse being released from the start chamber (B), checking a hole (C), and entering the escape tunnel (D).

      4. Following each trial, the maze and escape tunnel are cleaned with 70% ethanol.
      5. For each trial, a number of parameters are recorded to assess performance. These include the latency to locate (primary latency) and enter (total latency) the escape tunnel, along with the number of incorrect holes checked prior to locating (primary errors) and entering (total errors) the tunnel. Errors are defined as checking any hole that does not contain the escape tunnel and are scored live using a tally counter. The distance traveled (path length) prior to locating the escape tunnel and total distance for each trial are also chronicled. In addition, we also make note of the location of the first hole checked relative to the escape tunnel (primary hole distance) by a given mouse during each trial. For this measure, values range from 0 (target hole) to 20 (directly opposite the target hole). Finally, for each trial, the search strategy is classified as spatial, serial, or random (Figure 4). Trials where mice have scores of 3 or less for both primary errors and primary hole distance are defined as spatial searches (Video 1). Trials in which mice spent the majority of the time on the periphery performing systematic hole searches in a clockwise or counterclockwise manner are classified as a serial searches (Video 2). All other trials are considered random searches, including those in which mice failed to enter the escape tunnel within the 3-min trial period (Video 3).

        Figure 4. Barnes maze search strategy. A-C. Tracking data from individual trials that were classified as random (A), serial (B), and spatial searches (C).

        Video 1. Spatial search strategy

        Video 2. Serial search strategy

        Video 3. Random search strategy

    3. Probe trial
      1. Three days after the final session of acquisition training, mice undergo a 1 min probe trial in which the escape tunnel is removed from the apparatus.
      2. The probe trial is administered in a similar manner to the acquisition trials, except that the start chamber is placed in the center of the apparatus, rather than the center of any given quadrant.
      3. For the probe trial, the latency and distance traveled (path length) prior to reaching to previous escape tunnel location are recorded, along with the primary hole distance, total distance traveled, number of target hole checks, and number of incorrect holes checks.

Data analysis

  1. Under the ‘Mode’ tab in the VideoMot2 program, select ‘Analysis’.
  2. Under the ‘File’ tab, select the chosen file to be analyzed.
  3. Recorded data can be examined using the video player located in the lower right hand corner of the software program. Here the experimenter can determine and/or review the number of errors and target hole distance as well as classify the search strategy.
  4. Click on the ‘Protocol’ button situated in the lower right hand corner of the software program. This will open a new display that contains data for latency to target (primary latency) and distance traveled.
  5. After all relevant data for each trial is tabulated from VideoMot2, it is then consolidated into trial blocks using Microsoft Excel. Each trial block consists of four trials conducted over two consecutive days with one start location in each of the four quadrants. Mean values for each trial block are imported into GraphPad Prism 5.0 to generate graphs and to determine whether statistically significant differences exist between groups.
  6. Figure 5 displays graphs for measures of primary latency (A), primary errors (B), and primary hole distance (C) that compare groups of wild-type and knockout mice. Both groups showed similar decreases in all three measures with increased training, indicating that spatial learning occurred. Other Barnes maze parameters that are often graphed include path length, average speed, percentage of failed trials (3 min without finding escape tunnel), and percentage of trials using a spatial search strategy.

    Figure 5. Barnes maze data presentation. A-C. Graphs for measures of primary latency (A), primary errors (B), and primary hole distance (C) that compare performance between groups of wild-type and knockout mice. For primary hole distance, the dotted line indicates random chance performance (score = 10).


  1. One potential drawback of the Barnes maze is that the lack of stressful stimuli can result in slow learning. To provide mild stress and increase motivation for escape, we play 90 dB white noise through a loudspeaker during all trials. Other groups have used buzzer noise in a similar manner to induce escape behavior (Bach et al., 1995; O’Leary and Brown, 2012).
  2. It should also be noted that pharmacological and/or genetic manipulations that increase anxiety could act as confounding factors on Barnes maze performance. However, this potential confound appears to be of greater concern for the MWM than the Barnes maze. For example, one study used both the MWM and the Barnes maze to assess spatial learning in neurogranin null mice, a mutant strain with increased anxiety. These mice were unable to reach acquisition criterion on the MWM, but were able to do so for the Barnes maze (Miyakawa et al., 2001). Nevertheless, when testing mice with increased anxiety on the Barnes maze, decreasing the light intensity would be one possible remedy to minimize the confounding influence of anxiety.
  3. In addition to the procedure outlined above, we have also used a version with more extensive acquisition training that included 40 trials spread over 20 days (Pitts et al., 2013). In our studies, we have found that wild-type C57BL/6J mice develop a spatial preference after 15-20 acquisition trials, as indicated by primary hole distances significantly less than 10 (random chance). Further training results in decreasing primary hole distances and an increasing percentage of spatial search strategies. However, this additional training can also have a saturating effect, as cognitively impaired mice may eventually catch up with that of normal mice. There is no clear consensus among researchers as to when acquisition training should end. In the initial paper adapting the Barnes maze for use in mice, researchers kept testing a given mouse until it made 3 errors or less on 7 out of 8 consecutive trials (Bach et al., 1995). For wild-type mice used in this study, the median time to meet this criteria was 22 days on a training regimen of one trial daily. In the published literature since that time, the typical range is 15-30 acquisition trials (Seeger et al., 2004; Patil et al., 2009; O’Leary et al., 2011 and 2012) before administering the probe trial.
  4. Our experience with the Barnes maze is limited to the use of young (3-5 months of age) adult C57BL/6J mice. We have found that rates of learning can vary substantially between individual mice within a given group. Due to this variability, we recommend a group size of 10-12 mice (matched for age and gender) in order to accurately compare performance between groups on this task. In terms of gender differences, we have observed that males adopt a spatial search strategy to a greater degree than females as training progresses. Moreover, it also should be noted that other groups have reported significant differences in Barnes maze performance with respect to mouse background strain (O’Leary et al., 2011) and age (Kesby et al., 2015).
  5. Following administration of the probe trial, training can be further extended to examine reversal learning (Seeger et al., 2004). For reversal learning, the escape tunnel is moved to a location directly opposite the initial target site. Training proceeds in an analogous manner to that described above for initial acquisition and the same parameters are measured.


Our laboratory used this protocol to assess spatial learning in mice in two recent publications (Pitts et al., 2013 and 2015). That work was supported by NIH grants G12 MD007601, R01 DK47320 (Marla J. Berry), and Pilot Project Award funds from G12 MD007601 to M.W.P. The author thanks Ann Hashimoto, Ting Gong, Daniel Torres, and Tessi Sherrin for helping obtain the images and video that accompany this manuscript. The author declares that there are no any conflicting and/or competing interests.


  1. Bach, M. E., Hawkins, R. D., Osman, M., Kandel, E. R. and Mayford, M. (1995). Impairment of spatial but not contextual memory in CaMKII mutant mice with a selective loss of hippocampal LTP in the range of the theta frequency. Cell 81(6): 905-915.
  2. Barnes, C. A. (1979). Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat. J Comp Physiol Psychol 93(1): 74-104.
  3. Harrison, F. E., Hosseini, A. H. and McDonald, M. P. (2009). Endogenous anxiety and stress responses in water maze and Barnes maze spatial memory tasks. Behav Brain Res 198(1): 247-251.
  4. Kesby, J. P., Kim, J. J., Scadeng, M., Woods, G., Kado, D. M., Olefsky, J. M., Jeste, D. V., Achim, C. L. and Semenova, S. (2015). Spatial cognition in adult and aged mice exposed to a high-fat diet. PLoS One 10(10): e0140034.
  5. Iivonen, H., Nurminen, L., Harri, M., Tanila, H. and Puolivali, J. (2003). Hypothermia in mice tested in Morris water maze. Behav Brain Res 141(2): 207-213.
  6. Miyakawa, T., Yared, E., Pak, J. H., Huang, F. L., Huang, K. P. and Crawley, J. N. (2001). Neurogranin null mutant mice display performance deficits on spatial learning tasks with anxiety related components. Hippocampus 11(6): 763-75.
  7. Morris, R. (1984). Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11(1): 47-60.
  8. O’Leary, T. P. and Brown, R. E. (2012). The effects of apparatus design and test procedure on learning and memory performance of C57BL/6J mice on the Barnes maze. J Neurosci Methods 203(2): 315-324.
  9. O’Leary, T. P., Savoie, V. and Brown, R. E. (2011). Learning, memory and search strategies of inbred mouse strains with different visual abilities on the Barnes maze. Behav Brain Res 216(2): 531-42.
  10. Patil, S. S., Sunyer, B., Hoger, H. and Lubec, G. (2009). Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze, and the Morris water maze. Behav Brain Res 198(1): 58-68.
  11. Pitts, M. W., Kremer, P. M., Hashimoto, A. C., Torres, D. J., Byrns, C. N., Williams, C. S. and Berry, M. J. (2015). Competition between the brain and testes under selenium-compromised conditions: Insight into sex differences in Selenium metabolism and risk of neurodevelopmental disease. J Neurosci 35(46):15326-38.
  12. Pitts, M. W., Reeves, M. A., Hashimoto, A. C., Ogawa, A., Kremer, P., Seale, L. A. and Berry, M. J. (2013). Deletion of selenoprotein M leads to obesity without cognitive deficits. J Biol Chem 288(36): 26121-26134.
  13. Porsolt, R. D., Le Pichon, M. and Jalfre, M. (1977). Depression: a new animal model sensitive to antidepressant treatments. Nature 266(5604): 730-732.
  14. Seeger, T., Fedorova, I., Zheng, F., Miyakawa, T., Koustova, E., Gomeza, J., Basile, A. S., Alzheimer, C. and Wess, J. (2004). M2 muscarinic acetylcholine receptor knock-out mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity. J Neurosci 24(45): 10117-10127.


巴恩斯迷宫是一种基于干地的啮齿动物行为范式,用于评估空间学习和记忆,最初由同名的卡罗尔巴恩斯开发。 它代表了更受欢迎的莫里斯水迷宫的一个完善的替代方案,并提供了摆脱游泳行为的潜在混淆影响的优势。 在此,详细描述了巴恩斯迷宫实验装置和用于在小鼠中进行测试和分析的相应程序。

【背景】Barnes迷宫是一种基于干陆的行为测试,最初由Carol Barnes开发,用于研究老鼠的空间记忆(Barnes,1979),后来被改编用于老鼠(Bach et。,1995年)。从概念上讲,它类似于Morris水迷宫(Morris,1984),因为它是一个海马依赖的任务,动物学习周围环境远端线索与固定逃生位置之间的关系。对于老鼠,典型的巴恩斯迷宫装置包括一个高架的圆形平台,周围有40个均匀分布的孔。逃生通道安装在一个孔的下方,其余39个孔留空。明亮的光线和开放的空间都对啮齿动物厌恶,因此成为诱发逃避行为的动力因素。逃生通道在训练期间保持在固定位置,其中涉及多天的多次试验。在训练过程中,啮齿动物通常利用三种不同搜索策略(随机,序列,空间)的序列来了解逃生隧道的位置。在进行足够的采集训练后,逃生通道将被移除并进行探查试验以评估空间参考记忆。

尽管MWM是评估啮齿动物空间学习的主要模型,但Barnes迷宫提供了一些值得注意的重要优势。首先,巴恩斯迷宫不涉及游泳以及与之相关的潜在混杂因素。游泳是有压力的,详细研究表明,MWM训练使血浆皮质酮水平比巴恩斯迷宫更大程度地增加(Harrison等人,2009年)。此外,大多数MWM协议中使用的游泳条件引起核心体温降低,这可能会影响性能(Iivonen et al。 ,2003)。此外,啮齿动物经常采取漂浮,这被认为代表了行为绝望的状态,并被认为是广泛使用的Porsolt强迫游泳试验中的“抑郁样”行为的指标(Porsolt等人 ,1977)。最后,如上所述,巴恩斯迷宫允许在每次试验的执行过程中清楚描述鼠标使用的三种可能的搜索策略。

关键字:空间记忆, 老鼠, 海马, 认识, 行为


  1. 纸巾(Georgia-Pacific Consumer Products,目录号:48100)
  2. 70%乙醇喷雾瓶
  3. C57BL / 6J成年雄性小鼠(购自杰克逊实验室,3-5个月大)


  1. 光线充足(约1,000勒克斯)的测试室,附近有一个容纳室(图1A)
  2. Barnes迷宫装置(TSE Systems,目录号:302050-BM / M)包括:
    1. 包含40个等距孔(直径= 5厘米)的圆形PVC平台*(直径= 122厘米;厚度= 1厘米)(图1B)
    2. 灰色PVC起始室*由底板和盖子组成(图1C)
    3. PVC逃生通道*可安装在40个逃逸孔中的任何一个(图1D)
    4. 用于圆形PVC平台的铝支撑架*(高度= 80厘米)(图1E)
  3. 高架相机(松下,产品目录号:WV-BP332,图1F)

  4. 围绕平台的三个远端视觉提示(长度/宽度〜30厘米)(图1G)
  5. 用于90分贝白噪声的扬声器(索尼,目录号:SS-MB150H)
  6. 基于Windows的PC电脑(戴尔,型号:OptiPlex 780)连接到相机
  7. 理货柜台

    图1.巴恩斯迷宫实验装置。 :一种。用于分析的行为测试室和相邻房间的布局。 B-F。 Barnes迷宫平台(B),启动室(C),逃生通道(D),铝支架(E),头顶相机(F),单个视觉提示(G)的图像。


  1. TSE VideoMot2视频追踪软件(TSE Systems)
  2. GraphPad Prism版本5.0(GraphPad软件)
  3. Microsoft Excel


  1. 软件设置
    1. 校准
      1. 在VideoMot2软件程序左上角的“模式”选项卡下,选择“校准”(图2)。
      2. 点击“校准”按钮。
      3. 在Barnes迷宫平台的直径上绘制一条线。
      4. 在“真实长度”框中输入1220毫米。这对应于巴恩斯迷宫的实际直径。

        图2. VideoMot2分析软件的屏幕截图

    2. 定义实验区域
      1. 在VideoMot2程序的“模式”选项卡下,选择“实验区域”。
      2. 使用位于显示窗口底部的绘图工具制作一个覆盖整个Barnes平台的圆圈。这定义了软件将跟踪鼠标的区域。
    3. 定义信息区域
      1. 在VideoMot2程序的“模式”选项卡下,选择“信息区域”。

      2. 使用绘图工具在覆盖逃生通道的孔周围形成一个圆圈。
    4. 测量
      1. 从“模式”选项卡中选择“测量”。
      2. 在文字'Autostop at'右侧的白色方框中输入00:03:00。确保“Autostop at”文本左侧的白色框被选中。这将确保视频跟踪软件在3分钟后自动停止每次试用。
      3. 点击位于显示窗口右下角的“开始”按钮。

      4. 在提示时输入相关研究信息(研究编号,等。)。
      5. 单击一次空格键开始背景测量,然后再次结束。在背景测量过程中,确保照相机没有移动或照明变化。
      6. 提示时输入试验数据(动物编号,组)。
      7. 单击一次空格键开始跟踪鼠标。在视频显示中,十字会叠加在鼠标的身体上,并在迷宫中跟随它的运动。
      8. 要保存每个试用版的数据,请在屏幕左上角的“文件”选项卡下选择“另存为”。

  2. 巴恩斯迷宫程序(图3)
    1. 习惯(第1天)
      1. 将逃生通道连接到平台,并为每个小鼠习惯性试验添加一张干净的薄纸。周围的视觉线索应该到位。这些线索应该保持不变,以适应习惯,习惯和探索阶段。

      2. 将鼠标放置在逃生通道中1分钟
      3. 将鼠标放在设备的中心。让它探索,直到它进入逃生隧道或经过5分钟。

      4. 用70%乙醇清洁设备并逃离隧道
    2. 习得训练(第1-10天)
      1. 适应习惯和习得训练开始之间应至少间隔1小时。将逃生通道连接到与用于习惯性试验的位置不同的位置。在训练期间,逃生隧道的位置相对于房间中的空间线索保持在该固定位置。
      2. 训练包括每天两次采集试验(每次试验3分钟;间隔时间〜1小时),起始位置在四个象限中伪随机变化。
      3. 在每次试验开始时,将鼠标放置在位于四个象限中的一个的中心的灰色PVC开始室中。 15秒后,启动室被抬起(图3B),并允许鼠标探索迷宫。在每次试用期间,通过扬声器播放巨大的白噪声(90 dB)以诱导逃生行为。试验结束时,鼠标进入逃生通道(图3D)或3分钟过去。如果鼠标在3分钟内未能找到逃生通道,则由研究人员将其放置在通道中,并允许在移除之前停留15秒。

        图3. Barnes迷宫程序 A.实验时间表; B-d。从开始室(B)释放一只老鼠的图像,检查一个洞(C),然后进入逃生通道(D)。

      4. 每次试验后,迷宫和逃生隧道都用70%乙醇清洗。
      5. 对于每个试验,记录许多参数以评估性能。这些包括定位(主要延迟)和输入(总延迟)延迟,以及在定位(主要错误)和输入(总误差)隧道之前检查的错误孔数量。错误定义为检查任何不包含逃生通道的洞,并使用计数器计数器进行实时评分。还记录了定位逃生隧道之前的行进距离(路径长度)和每次试验的总距离。另外,我们还记录了每次试验期间给定鼠标相对于逃生隧道检查的第一个孔的位置(主孔距离)。对于此测量,值范围从0(目标孔)到20(与目标孔正对)。最后,对于每个试验,搜索策略被分类为空间,连续或随机(图4)。将小鼠的主要误差和主要洞距均小于或等于3分的试验定义为空间搜索(视频1)。小鼠大部分时间在外围进行的试验以顺时针或逆时针方式进行系统孔搜索,被分类为连续搜索(视频2)。所有其他试验被认为是随机搜索,包括在3分钟试用期内没有进入逃生通道的那些试验(视频3)。

        图4. Barnes迷宫搜索策略。 A-C。跟踪分类为随机(A),连续(B)和空间搜索(C)的单独试验的数据。




    3. 探测试验
      1. 在最后一次采集训练后的三天,小鼠进行1分钟的探针试验,其中逃生通道从设备中移除。
      2. 除了将起始室放置在设备的中心而不是任何给定象限的中心之外,探测试验以与采集试验相似的方式进行。
      3. 对于探测试验,记录到达前一个逃生隧道位置之前的行程延迟和距离(路径长度),以及主孔距离,总行程距离,目标孔检查次数和不正确的孔检查次数。 br />


  1. 在VideoMot2程序的“模式”选项卡下,选择“分析”。
  2. 在“文件”选项卡下,选择要分析的选定文件。
  3. 录制的数据可以使用位于软件程序右下角的视频播放器进行检查。在这里,实验者可以确定和/或审查错误的数量和目标洞距,并对搜索策略进行分类。
  4. 点击位于软件程序右下角的“协议”按钮。这将打开一个新的显示,其中包含延迟目标(主要延迟)和行进距离的数据。
  5. 在每个试验的所有相关数据从VideoMot2列表后,然后使用Microsoft Excel将其合并为试用区块。每个试验区由连续两天进行的四项试验组成,每个试验区在四个象限中各有一个起始位置。将每个试验块的平均值输入到GraphPad Prism 5.0中以生成图表并确定组间是否存在统计学显着差异。
  6. 图5显示比较野生型和敲除小鼠组的主要潜伏期(A),主要错误(B)和主要洞距(C)的图。两组均显示类似的减少,所有三项措施都增加了培训,表明发生了空间学习。其他Barnes迷宫参数通常包括路径长度,平均速度,失败试验的百分比(3分钟没有发现逃生隧道)以及使用空间搜索策略的试验百分比。

    图5.巴恩斯迷宫数据展示。 A-C。主要潜伏期(A),主要误差(B)和主孔距离(C)的比较图,比较野生型和敲除小鼠组之间的表现。对于主洞距离,虚线表示随机机会表现(得分= 10)。


  1. 巴恩斯迷宫的一个潜在缺陷是缺乏压力刺激会导致学习缓慢。为了提供轻微的压力并增加逃生动机,我们在所有试验中通过扬声器播放90 dB白噪声。其他团体以相似的方式使用蜂鸣器噪音来诱导逃跑行为(Bach et。,1995; O'Leary and Brown,2012)。
  2. 还应该指出,增加焦虑的药理学和/或遗传操作可以作为Barnes迷宫表现的混杂因素。然而,这种潜在的混乱似乎是MWM比巴恩斯迷宫更为关注的问题。例如,一项研究使用MWM和Barnes迷宫来评估神经颗粒素无效小鼠(一种焦虑增加的突变株)的空间学习。这些小鼠无法达到MWM上的采集标准,但能够为Barnes迷宫(Miyakawa等人,2001)做到这一点。尽管如此,在巴恩斯迷宫中测试焦虑增加的小鼠时,减少光强度将是减少焦虑影响的一种可能的补救措施。
  3. 除了上面列出的程序外,我们还使用了一个更广泛的采集培训版本,其中包括20天内发布的40个试验(Pitts et。 ,2013)。在我们的研究中,我们发现野生型C57BL / 6J小鼠在15-20次采集试验后发展空间偏好,如主孔距明显小于10(随机几率)所示。进一步的培训会减少主孔距离,并增加空间搜索策略的比例。然而,这种额外的训练也可以具有饱和效应,因为认知障碍的老鼠可能最终赶上正常老鼠。研究人员之间没有明确的一致意见,何时应该结束培训。在最初的文章中,巴恩斯迷宫适用于小鼠,研究人员不断对一只给定的小鼠进行测试,直到它在8次连续试验中的7次出现3次或更少的错误(Bach et。,1995年)。对于本研究中使用的野生型小鼠,每天进行一次试验的培训方案中,符合此标准的中位时间为22天。从那时起,在公开的文献中,典型的范围是15-30次采集试验(Seeger等人,2004; Patil等人,2009; O'Leary等人,等,2011和2012)。
  4. 我们对巴恩斯迷宫的经验仅限于使用年轻(3-5个月大)的成年C57BL / 6J小鼠。我们发现,给定组内的个体小鼠之间的学习速度可能有很大差异。由于这种差异,我们建议将10-12只小鼠(年龄和性别匹配)的小组大小为准确比较这项任务之间的群体之间的表现。在性别差异方面,我们观察到,随着培训的进展,男性采用空间搜索策略的程度高于女性。此外,还应该指出的是,其他组已经报道了在小鼠背景应变(O'Leary等人,2011)和年龄(Kesby等人)方面Barnes迷宫表现的显着差异。,2015)。
  5. 在进行探查试验之后,可以进一步延伸训练以检查逆转学习(Seeger等人,2004年)。为了反向学习,逃生通道移动到与最初目标站点正对的位置。培训以与上述初始采集类似的方式进行,并测量相同的参数。


我们的实验室在最近的两个出版物中使用该协议来评估小鼠的空间学习(Pitts等人,2013和2015)。这项工作得到了美国国立卫生研究院拨款G12 MD007601,R01 DK47320(Marla J. Berry)的支持,以及G12 MD007601向M.W.P提供的试点项目奖金。作者感谢Ann Hashimoto,Ting Gong,Daniel Torres和Tessi Sherrin帮助获取伴随此稿件的图像和视频。作者声明,没有任何冲突和/或竞争利益。


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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Pitts, M. W. (2018). Barnes Maze Procedure for Spatial Learning and Memory in Mice. Bio-protocol 8(5): e2744. DOI: 10.21769/BioProtoc.2744.
  2. Pitts, M. W., Kremer, P. M., Hashimoto, A. C., Torres, D. J., Byrns, C. N., Williams, C. S. and Berry, M. J. (2015). Competition between the brain and testes under selenium-compromised conditions: Insight into sex differences in Selenium metabolism and risk of neurodevelopmental disease. J Neurosci 35(46):15326-38.