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Social Observation Task in a Linear Maze for Rats
大鼠线性迷宫社会观察任务实验   

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Abstract

Animals often learn through observing their conspecifics. However, the mechanisms of them obtaining useful knowledge during observation are beginning to be understood. This protocol describes a novel social observation task to test the ‘local enhancement theory’, which proposes that presence of social subjects in an environment facilitates one’s understanding of the environments. By combining behavior test and in vivo electrophysiological recording, we found that social observation can facilitate the observer’s spatial representation of an unexplored environment. The task protocol was published in Mou and Ji, 2016.

Keywords: Hippocampus(海马), Place cell(位置细胞), Social observation(社会观察), Local enhancement(局部增强)

Background

Social learning is defined as acquiring new knowledge through observing or interacting with others (Heyes and Galef, 1996; Bandura, 1997; Meltzoff et al., 2009). One form of social learning utilized by many species is the so-called ‘local enhancement’ (Heyes and Galef, 1996): an animal’s understanding of an environment is facilitated by the presence of other social subjects in the same environment. Animals achieve local enhancement possibly by heightened attention, acquiring environmental attributes such as safety or food availability, or other unspecified means (Zajonc, 1965; Heyes and Galef, 1996; Zentall, 2006). The hypothesis predicts that the presence of social subjects in an environment impacts other animals’ neural processing of information related to the environment, therefore facilitate their understanding of the environment.

It has been shown that spatial information of an environment is represented by hippocampal place cells (O’Keefe and Dostrovsky, 1971; Wilson and McNaughton, 1993; Burgess and O’Keefe, 2003) in rodents and humans. Place cells become active at specific locations of a given environment, called place fields. We asked how an observer’s place cell sequence representing an environment can be influenced by another rat navigating in the environment, even if the observer is located in a physically different environment. This protocol is designed to explore the neural basis of such local enhancement effect of social observation. Specifically, we monitored the hippocampus place cells in observer rats as they stayed in a small box while a demonstrator rat was running on a separate, nearby linear track, and then later when observer rats were running the same track themselves. Our results show that observer’s place cell sequences during track running also appeared in the box during observation, but only when a demonstrator was present on the track. Observer’s running speed, number of run laps and place cells’ specificity are significantly higher than those in control animals.

Materials and Reagents

  1. 3-6 month old male Long Evans rats, 450-550 g
  2. 4x diluted sweetened condensed milk (Eagle Brand) are used for reward
  3. 70% ethanol for cleaning the maze between daily training sessions

Equipment

  1. A 2-m long linear track made of galvanized steel (Figures 1A and 1B)
  2. A small 25 (length) x 25 (width) x 40 (height) cm box. Three sides of the box has opaque, high (40 cm) walls, leaving only one side open toward the track (Figure 1C)
  3. Milk wells located at the two ends of the linear track. Milk reward was remotely delivered by syringe and tubing from behind a curtain
  4. Curtain separating the experimenter and recording setup (Figure 1D)
  5. A 60 (length) x 60 (width) x 100 (height) cm rest box. The rest box was placed ~1 m away from the track. Animals were placed in a ceramic plate (20 cm in diameter) on top of a 30-cm tall flower pot, located at the center of the enclosed rest box for resting (Figure 1E)
  6. The animal’s position was tracked by red and green LED mounted over on the head. Position data were recorded by a ceiling camera at 33 Hz
  7. In vivo extracellular recording equipment is described earlier in Mou and Ji, 2016. Tetrode recording was made by a Digital Lynx acquisition system (Neuralynx, model: Digital Lynx Acquisition System ). Spikes from single neurons were sampled at 32 kHz and online-filtered between 600 Hz and 9 kHz. Local field potentials (LFPs) were sampled at 2 kHz and online-filtered between 0.1-1 kHz


    Figure 1. Social observation apparatus. A. Schematic depiction of the recording setting; B. Linear track; C. Observation box; D. Curtain; E. Flower pot in a rest box.

Software

  1. Customized MATLAB script

Procedure

Note: All experimental procedures followed the guidelines by the National Institute of Health and were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.

  1. 3-6 month-old male Long Evans rats, weighted between 450 g to 550 g, are housed 2-3 per cage and handled daily for ~7 days.
  2. After the acclimation period, demonstrator rats are food restricted to 85-90% of their baseline body weight before experiment. In the meantime, they are trained to run the linear track back and forth for milk reward for at least one week. Training session takes place once a day and lasted about 20 min. An adult rat can achieve satisfactory behavioral performance (continuous run back and forth without prolonged pause) within 2-3 days.
  3. While demonstrator rats are being trained, non-trained naïve rats are implanted with hyperdrive that contains 15 independently movable tetrodes and one reference electrode. Tetrodes target the right dorsal hippocampal CA1 region (coordinates: anteroposterior -3.8 mm, mediolateral 2.4 mm relative to bregma). During surgery, all tetrodes are placed right above the surface of exposed brain tissue without touching it. Then tetrodes are advanced individually to reach dorsal CA1 area. Since the dorsal CA1 area is not flat, the final depths vary among individual tetrodes, but approximately 2 mm below the dura. The reference tetrode is placed in white matter above dorsal CA1 pyramidal cell layer, approximately 1.7 mm below the dura.
  4. After having fully recovered from surgery (typically within 3 days), the implanted rats are food deprived to 85-90% of their baseline weight.
  5. During the following 3-4 weeks after implantation, tetrodes are slowly advanced to the CA1 pyramidal layer until sharp-wave ripples signal were observed (Figure 2). The reference tetrode is placed in the white matter above the CA1. Recordings are not conducted within 24 h after tetrodes movement.


    Figure 2. A representative LFP sharp-wave ripple

  6. Pre-recording training phase: Each rat is placed in the observation box for 2 or 3 days, 15-30 min each day. For a group of implanted rats, there is a well-trained demonstrator running on the track in this pre-recording phase. For the other group of implanted rats, the track is left empty without a demonstrator.
  7. Recording procedure: The recording starts and lasts for 6-12 consecutive days after pre-recording training. The recording procedure is depicted in Figure 3. A typical recording took ~2 h. Each recording consists of three sessions.


    Figure 3. Schematic of daily recording procedure. The Pre- and Post-box sessions were configured with various conditions.

    For the group of rats that have watched a well-trained demonstrator in the pre-recording training phase, on the first recording day:
    1. The implanted rats first stay in the observation box while a well-trained demonstrator is running the linear track for 15 min (Pre-box session).
    2. Then the implanted rat runs the linear track for the first time (Track session).
    3. Then the implanted rat stays in the observation box again while the well-trained demonstrator is running the track (Post-box session).

      Note: The recorded rats have never been exposed to the track before the first recording day.

      In each of the following days, the Pre-box and Post-box session is set up in various ways as the following, while the Track session remains the same. Each condition is recorded for 1-3 days.

      1. Empty-track: removing the demonstrator from the track.
      2. No-track: removing both the track and demonstrator.
      3. Naïve-demo: replacing the demonstrator with a naïve demonstrator that have never been exposed to the track.
      4. Toy-car: replacing the demonstrator with a toy car remotely controlled by the experimenter behind the curtain. The car is maneuvered to move at a speed comparable to a rat’s. The toy car stops when it reaches the end of the track then reverses its direction.
      5. Blocked-view: implanted rats stay in the box but with the view blocked while a well-trained demonstrator is running on the track. In this condition, the observation box is rotated 180° such that the opening side now is facing a nearby wall of the room 20 cm away. The implanted rat in the box can not see either the track or the demonstrator, but has access to the auditory and olfactory information associated with the demonstrator.

        For the other group of rats that have seen only the empty track in the pre-recording training, the Pre-box and Post-box sessions on the first day are under Empty-track condition. In the following days, the Pre-box and Post-box sessions are replaced by Trained-demo and other conditions as described above.

Data analysis

  1. We tracked animals’ positions using the red and green LED mounted on their heads. Position data were recorded by a ceiling camera and analyzed off-line using customized MATLAB script. The observer rats’ behavior in the Track session was quantified by mean running speed and number of running laps per trajectory (one running direction, Figure 4A). Our data show that both parameters in the observer rats after watching the well-trained demonstrator were significantly greater than those watching the empty track during pre-recording training phase.
  2. All extracellular recording data, including spike timestamps, LFPs, were digitized and analyzed off-line using customized MATLAB routines (MATLAB routines are available upon reasonable request). After watching well-trained demonstrator, observer rats’ place cell firing appeared to be less dispersed than those watching Empty-track. Place cells’ firing sparsity measured by spatial information was significantly greater in the former group (Figure 4B). Taken together, our data suggest an improvement in understanding of a novel environment measured by track-running performance and place field development. Detailed analysis can be found in Mou and Ji, 2016.


    Figure 4. Experience in the box improved behavioral performance and novel place field development on the track. A. Mean running speed and number of laps per trajectory in Track sessions of first two days (Day1, Day2) for the Trained-demo (N = 9) and Empty-track (N = 5) rats; B. Spatial information for all active cells under each condition (Trained-demo, Empty-track) on each day (Day1, Day2). Number on top of each bar: the number of cells. There was a significant main effect between the conditions (F(1,791) = 21, p = 0, Two-way ANOVA), but not between days (F(1,791) = 1.4, p = 0.25). Reproduced from Mou and Ji, 2016 with permission.

Acknowledgments

The authors would like to thank the entire Ji lab for help on constructing and configuring the apparatus, and for suggestions on preparation of the manuscript. This work was supported by grants NIMH R01MH106552, Simons Foundation 273886 to D.J.

References

  1. Bandura, A. (1997). Social learning theory. General Learning Press.
  2. Burgess, N. and O’Keefe, J. (2003). Neural representations in human spatial memory. Trends Cogn Sci 7(12): 517-519.
  3. Heyes, C. M. and Galef Jr, B. G. (1996). Social learning in animals: the root of culture. Academic Press.
  4. Meltzoff, A. N., Kuhl, P. K., Movellan, J. and Sejnowski, T. J. (2009). Foundations for a new science of learning. Science 325(5938): 284-288.
  5. Mou, X and Ji, D. (2016). Social observation enhances cross-environment activation of hippocampal place cell patterns. eLife 5: e18022.
  6. O’Keefe, J. and Dostrovsky, J. (1971). The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res 34(1):171-175.
  7. Wilson, M. A. and McNaughton, B. L. (1993). Dynamics of the hippocampal ensemble code for space. Science 261(5124): 1055-1058.
  8. Zajonc, R. B. (1965). Social facilitation. Science 149(3681): 269-274.
  9. Zentall, T. R. (2006). Imitation: definitions, evidence, and mechanisms. Anim Cogn 9(4): 335-353.

简介

动物经常通过观察他们的特异性来学习。 然而,他们在观察期间获得有用知识的机制开始被理解。 该协议描述了一种新的社会观察任务,用于测试“局部增强理论”,其中提出在环境中存在社会主体有助于人们了解环境。 通过结合行为测试和体内电生理记录,我们发现社会观察可以促进观察者对未开发环境的空间表示。 任务协议于2016年在Mou和Ji发布。
【背景】社会学习被定义为通过观察或与他人互动来获取新知识(Heyes and Galef,1996; Bandura,1997; Meltzoff等人,2009)。许多物种利用的一种社会学习形式是所谓的“地方增强”(Heyes和Galef,1996):动物对环境的理解由同一环境中其他社会科目的存在所促进。动物可能通过加强注意力获得当地的增强,获取环境属性,如安全性或食物可获得性或其他未指定的手段(Zajonc,1965; Heyes和Galef,1996; Zentall,2006)。假设预测社会科目在环境中的存在会影响其他动物对与环境有关的信息的神经处理,从而促进他们对环境的理解。
已经表明,环境的空间信息由啮齿动物和人类的海马位置细胞(O'Keefe和Dostrovsky,1971; Wilson和McNaughton,1993; Burgess和O'Keefe,2003)表示。放置单元格在给定环境的特定位置(称为放置字段)处于活动状态。我们询问,即使观察者位于物理上不同的环境中,表示环境的观察者位置单元格序列也可能受到在环境中导航的另一个鼠标的影响。该协议旨在探索社会观察这种局部增强效应的神经基础。具体来说,当观察者大鼠停留在小盒子中时,我们监测观察者大鼠中的海马位置细胞,而示范性大鼠在独立的附近的线性轨道上运行,然后在观察者大鼠自己运行相同的轨道时。我们的研究结果表明,轨道运行期间观察者的位置细胞序列也在观察期间出现在框中,但只有当演示者存在于轨道上时。观察者的运行速度,运行次数和细胞的特异性显着高于对照动物。

关键字:海马, 位置细胞, 社会观察, 局部增强

材料和试剂

  1. 3-6个月大的男性Long Evans大鼠,450-550克
  2. 4倍稀释的加浓炼乳(Eagle Brand)用于报酬
  3. 70%乙醇用于清洁日常训练之间的迷宫

设备

  1. 2米长的镀锌钢线轨迹(图1A和1B)
  2. 小25(长)×25(宽)×40(高)厘米)的盒子。箱子的三面具有不透明,高(40厘米)的墙壁,只有一侧朝向轨道开放(图1C)
  3. 位于线性轨道两端的牛奶井。牛奶奖励通过注射器和管道从窗帘后面远程传送
  4. 分离实验者和录音设置的窗帘(图1D)
  5. 一个60(长)×60(宽)×100(高)厘米的休息盒。休息箱被放置在离轨道约1米处。将动物放置在位于封闭式休息箱中心的30厘米高的花盆顶部的陶瓷板(直径20厘米)中(图1E)
  6. 动物的位置被红色和绿色的LED跟踪在头上。位置数据由天花板相机以33 Hz记录
  7. 细胞外记录设备早在Mou和Ji于2016年描述。Tetrode记录由Digital Lynx采集系统(Neuralynx,型号:Digital Lynx Acquisition System)制成。来自单个神经元的峰值以32kHz采样,并在600Hz和9kHz之间进行在线滤波。本地场电位(LFP)以2 kHz采样,并在0.1-1 kHz之间进行在线滤波

    图1.社会观察设备。 A.录音设置的示意图;线性轨迹观察箱;窗帘E.在一个休息盒里的花盆。

软件

  1. 定制MATLAB脚本

程序

注意:所有实验程序均遵循国家卫生研究院的指导方针,并获得贝勒医学院机构动物护理与使用委员会的批准。

  1. 3-6个月大的雄性长伊万斯大鼠,重量在450g至550g之间,每笼笼养2-3次,每天处理约7天。
  2. 适应期后,实验前示范大鼠食物限于基线体重的85-90%。在此期间,他们经过训练,可以运行线性轨迹来回送牛奶,至少一周。培训课程每天进行一次,持续时间约为20分钟。成年大鼠可以在2-3天内达到令人满意的行为表现(连续不间断地进行而不间断地延续)
  3. 在示范老鼠正在接受训练时,未经训练的幼稚鼠被植入包含15个独立可移动的四极杆和一个参比电极的超速驱动。 Tetrodes针对右侧背侧海马CA1区域(坐标:前后-3.8 mm,相对于宝石的内侧2.4 mm)。在手术过程中,所有tetrodes都放置在暴露的脑组织表面的正上方,而不用接触它。然后四肢先进先进到达背部CA1区域。由于背部CA1区域不平坦,最终的深度在各个tetrodes之间变化,而在硬脑膜下约2mm。参考四极放置在背侧CA1锥体细胞层上方的白质中,硬膜下方约1.7 mm。
  4. 在手术完全恢复(通常在3天内)后,植入的大鼠被剥夺了基线重量的85-90%。
  5. 在植入后的3-4周期间,四面体缓慢前进到CA1锥体层,直到观察到尖波波纹信号(图2)。参考四极杆放置在CA1上方的白色物质中。录音不得在四弦琴运动后24小时内进行。

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    图2.代表性的LFP清晰波纹波纹

  6. 预记录训练阶段:每只大鼠每天在观察箱中放置2或3天,15-30分钟。对于一组植入的大鼠,在这个预录制阶段,有一个训练有素的示威者在轨道上运行。对于另一组植入的大鼠,轨道在没有示威者的情况下为空。
  7. 录音程序:预录录后录音开始持续6-12天。记录过程如图3所示。典型的记录需要〜2小时。每个录音由三个会话组成。


    图3.日记录程序示意图。 前后会话配置有各种条件。

    对于在录音前培训阶段观察训练有素的示威者的老鼠,在第一个录音日:
    1. 植入的大鼠首先停留在观察盒中,而训练有素的演示者正在运行线性轨迹15分钟(预制会话)。
    2. 然后植入的大鼠第一次运行线性轨迹(轨道会话)。
    3. 然后植入的大鼠再次停留在观察箱中,而训练有素的演示者正在运行轨道(邮箱会话)。

      注意:记录的老鼠在第一个录音日之前从未暴露在轨道上。

      在接下来的每一天,Pre-Box和Post-Box会话以各种方式设置如下,而Track会话保持不变。每个条件记录1-3天。

      1. 空轨道:从轨道上移除示威者。
      2. 无轨道:移除轨道和示威者。
      3. 天真的演示:用一个从未暴露在轨道上的天真示威者取代示威者。
      4. 玩具车:用幕后实验者远程控制的玩具车代替示威者。这辆车是以与老鼠相当的速度来操纵的。玩具车到达轨道末端时停止,然后反转方向。
      5. 封闭视图:植入的大鼠留在盒子里,但是在训练有素的示威者在轨道上运行时,阻挡了视野。在这种情况下,观察盒旋转180°,使得开口侧现在面向距离房间20cm的附近的墙壁。盒子中的植入的大鼠不能看到轨道或示威者,但是可以访问与示威者相关的听觉和嗅觉信息。

        对于在录音前训练中只看到空轨的另一组老鼠,第一天的前箱和邮箱会话都处于空轨状态。在接下来的日子里,前面的盒子和邮箱的会话被如上所述的训练演示和其他条件所取代。

数据分析

  1. 我们用红色和绿色的LED跟踪动物的位置。位置数据由天花板摄像头记录,并使用定制的MATLAB脚本离线分析。观察者大鼠在轨道会话中的行为通过平均运行速度和每个轨迹的运行圈数(一个运行方向,图4A)进行量化。我们的数据显示观察老鼠观察训练有素的示威者后的两个参数都明显大于在录音前训练期间观察空轨的参数。
  2. 所有细胞外记录数据(包括峰值时间戳,LFP)都使用定制的MATLAB例程进行数字化和离线分析(MATLAB例程可在合理要求下提供)。在观察训练有素的演示者之后,观察者大鼠的放置细胞发射似乎比那些观察空轨的人分散。在前一组中,通过空间信息测量的放置细胞的发射稀疏度显着更大(图4B)。总而言之,我们的数据表明,通过追踪运行绩效和场地开发来衡量的新环境的理解有所改善。详细分析可以在Mou和Ji,2016中找到。


    图4.框中的经验改进了行为表现和新的场地开发轨迹。 A.前两天(第1天,第2天)的轨道会话中每个轨迹的平均运行速度和圈数训练演示(N= 9)和空轨(N= 5)大鼠; B.每天(Day1,Day2)下每个条件(训练演示,空轨)下的所有活动细胞的空间信息。每个栏顶部的数字:单元格数。条件(F(1,791)= 21,p= 0,双向方差分析)之间存在显着的主要影响,但不是在日期之间(F(1,791)

致谢

作者要感谢整个吉实验室的建造和配置设备方面的帮助,以及有关准备稿件的建议。这项工作得到了NIMH R01MH106552,Simons Foundation 273886至D.J.的支持。

参考

  1. Bandura,A。(1997)。社会学习理论。普通学习新闻。
  2. Burgess,N.和O'Keefe,J.(2003)。人类空间记忆中的神经表示。趋势科技<7>(12):517-519。
  3. Heyes,CM和Galef Jr,BG(1996)。&lt; a class=“ke-insertfile”href=“https://books.google.com.tw/books/about/Social_Learning_In_Animals.html?id=Bp_xLfDBV8AC&redir_esc= y”target=“_ blank”>动物社会学习:文化的根源 学术出版社。
  4. Meltzoff,AN,Kuhl,PK,Movellan,J.和Sejnowski,TJ(2009)。&lt; a class=“ke-insertfile”href=“https://www.ncbi.nlm.nih.gov/pubmed/ 19608908”target=“_ blank”>新的学习科学基础。 科学 325(5938):284-288。
  5. Mou,X和Ji,D。(2016)。社会观察增强了海马位置细胞模式的跨环境激活。 eLife 5:e18022。
  6. O'Keefe,J.和Dostrovsky,J.(1971)。 海马作为空间地图。自由移动的大鼠的单位活动的初步证据。
    Brain Res 34(1):171-175。
  7. Wilson,MA和McNaughton,BL(1993)。&nbsp; 动力学海洋合奏代码的空间。 科学 261(5124):1055-1058。
  8. Zajonc,RB(1965)。&lt; a class=“ke-insertfile”href=“http://www.psychwiki.com/wiki/Zajonc,_R._B._(1965)._Social_facilitation._Science,_149, _269-274”。目标=“_ blank”>社会便利化。 科学 149(3681):269-274。
  9. Zentall,TR(2006)。&nbsp; 模仿:定义,证据,和机制。动画认知 9(4):335-353。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mou, X. and Ji, D. (2017). Social Observation Task in a Linear Maze for Rats. Bio-protocol 7(13): e2361. DOI: 10.21769/BioProtoc.2361.
  2. Mou, X and Ji, D. (2016). Social observation enhances cross-environment activation of hippocampal place cell patterns. eLife 5: e18022.
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