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Testing Depression in Mice: a Chronic Social Defeat Stress Model
小鼠抑郁症检测:慢性社会挫败应激模型   

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Abstract

A vast challenge within neuropsychiatric research has been the development of animal models that accurately reflect symptoms associated with affective disorders. An ethologically valid model that has been shown to be effective in studying depression is the chronic social defeat stress model. In this model, C57BL/6J mice are subjected to chronic social defeat stress induced by CD-1 aggressor mice for 10 consecutive days. Discussed here is a protocol describing the screening process of the CD-1 aggressor mice, the confrontations between the C57BL/6J and CD-1 aggressor mice, and analysis of social avoidance scores as an indication of depression-like behaviors.

Keywords: Social defeat(社会挫败), Depression(抑郁症), Chronic stress(慢性应激), Social interaction(社会互动), Mouse model(小鼠模型)

Background

Depressive disorders are recurring and life-threatening conditions that affect approximately 120 million people worldwide. Of significant interest within the field of neuropsychiatric research is the development of valid animal models for depression to aid the understanding of the neurobiological and molecular mechanisms underlying depressive disorders. Various models of chronic stress have been used to induce symptoms in mice that are relevant to depression, among those are chronic unpredictable stress, foot-shock stress, immobilization stress followed by measurements of anhedonia or despair-like behaviors (e.g., sucrose preference test, forced swim test and tail suspension test), but they are not well validated as models of human depression (Krishnan and Nestler, 2011). Currently, chronic social defeat stress has been widely accepted as a well-validated model for depression, because of its reliability and reproducibility (Berton et al., 2006; Krishnan et al., 2007; Golden et al., 2011; Krishnan and Nestler, 2011; Russo and Nestler, 2013). The social defeat paradigm has been validated as a good model of human depression because of following reasons: 1) The depression phenotypes of susceptible mice (social avoidance, anhedonia, metabolic changes, etc.) are reversed by chronic treatment of anti-depressant drugs but not by acute administration. 2) Social defeat stress produces both a susceptible and resilient phenotype and this can be used to explain individual differences of human stress susceptibility in depression pathophysiology. 3) The susceptible phenotypes induced by social defeat stress are long lasting with concomitant genetic and epigenetic changes in the brain reward circuitry.

Materials and Reagents

  1. C57BL/6J (THE JACKSON LABORATORY): 7-8 weeks old mice are used in social defeat (6-7 weeks mice are ordered for a week acclimation period in the vivarium)
    Note: Mice are housed in standard cages with 4 to 5 mice per cage.
  2. CD-1 mice (Charles River): Retired male breeder mice were 4-6 months of age and singly housed in standard mouse cages
    Note: Social defeat paradigms use a consistent subordination of subject mice as a social stressor and a forced subordination strategy is employed in this protocol with the most widely used strains, C57BL/6J: CD-1 pair.
  3. Cleaning solution (for cleaning arena and custom Plexiglas social interaction arena/divider in between trials): 0.5% hydrogen peroxide

Equipment

  1. Clear rat cages (disposable rat cage with bedding) (Innovive, catalog number: RS1-H-C8 )
  2. Standard water bottle (mouse water bottle) (Innovive, catalog number: M-WB-300 )
  3. Water bottle stopper with sipper tube (Neoprene stopper, size: #8.5; Ancare, 1” bend tubes – open tip) (Ancare, catalog number: OT-199 - 3” )
  4. Cylinder (custom made, plastic holder for additional water spout; Figure 2B)
  5. Divider (custom made, clear perforated Plexiglas plate; Figure 2B)
  6. Removable enclosure (custom made, clear Plexiglas, top and bottom sides are open; Figure 3B)
  7. Stopwatch
  8. Open field chamber (custom made, 44 [w] x 44 [d] x 30 [h] cm)
  9. Video tracking system for social interaction test (e.g., Noldus Information Technology, model: EthoVision system , CCD camera & computer)

Software

  1. Tracking software: EthoVision XT (Noldus Information Technology)

Procedure

Note: Chronic social defeat stress experiments must comply with governmental and institutional guidelines for care and use of laboratory animals.

  1. Screening of CD-1 aggressor mice
    1. Order male CD-1 retired breeder mice at 4-6 months of age (order at least 2-3 times more than required, as less than half of CD-1 mice will be aggressive enough). CD-1 mice should be singly housed in standard mouse cages with access to food and water ad libitum. Allow CD-1 mice to habituate to their new environment for a week prior to initiating screening process.
    2. C57BL/6J ‘screener’ male mice are used only to screen CD-1 mice. The screener mice can range in age (8-20 weeks) and can be used for subsequent screening processes. Remove any aggressive ‘screener’ mice (rare).
    3. Screening of CD-1 mice is performed in the cage of the resident CD-1 mouse. C57BL/6J ‘screener’ mouse should be placed directly into home cage with aggressor CD-1 mouse for 5 min. Latency to aggression should be noted for each CD-1. Perform three screening sessions for 3 consecutive days (once daily) with different screeners.
    4. CD-1 aggressor mice should be selected based on two criteria: During three 5-min screening sessions, the latency to initial aggression must be less than 60 sec during two consecutive sessions. And the CD-1 must continuously attack the C57BL/6J screener mouse for at least 10 sec without interruption for all the three 5-min sessions.
    5. House CD-1 aggressor mice selected for experimentation with access to food and water ad libitum (ready to use). Aggressor mice can be used for up to 3 months after initial screening (CD-1 aggressor mice can habituate to social defeat sessions, and thus need to be rescreened prior to the next social defeat experiment).
  2. Chronic social defeat stress (Figure 1)


    Figure 1. Schematic of social defeat and social interaction test. Chronic social defeat scheme uses 10-days of consecutive social defeat using CD-1 aggressor mice in the divided cages, followed by social interaction test and subsequent analysis.

    1. To assemble social defeat cages (Figure 2A), take standard rat cage and fill the bottom of cage with woodchip bedding. Place the perforated Plexiglas divider in the center of cage. Divider will physically separate the mice after defeat sessions and allow for sensory interaction during non-defeat housing periods. Place cylinder (additional water bottle holder) on side opposite of standard water bottle side. Insert assembled water bottle with cylindrical holder. Ensure that water is easily accessible. Place food on bottom of each side (water and food should be available ad libitum). Place lid of standard rat cage securely onto cage. At least 10 social defeat cages with CD-1 aggressors are needed for the 10-day social defeat protocol.
      Note: The divider should be firmly positioned to prevent escapes.


      Figure 2. Standard cage setup in chronic social defeat stress. A. Standard rat cage used in chronic social defeat stress sessions. The resident aggressor CD-1 mouse is permanently housed on one side of the perforated divider, while C57BL/6J mice are rotated each day following defeat session to avoid habituation to aggressor mouse. B. Dimensions of additional parts for standard rat cage (custom made). The Cylinder holds an additional water bottle on side opposite of standard water bottle side. The perforated Plexiglas divider physically separates mice during non-social defeat periods (perforations allow for sensory interaction).

    2. Place resident CD-1 aggressor mouse on one side of the divided home cage overnight prior to the start of social defeat sessions for habituation in the new cage.
      Note: The cylinder side is preferred for efficient defeats in smaller space.
    3. On the first day of social defeat sessions, place intruder C57BL/6J mouse directly into the cage side of CD-1 aggressor mouse. After 5-10 min of social defeat (Video 1), transfer the intruder mouse to opposite side of perforated divider and house within compartment for the remainder of the 24-h period.
      Note: Placing intruder mouse close to the aggressor at session start is helpful to initiate aggressive behaviors. Social defeat stress may be done with up to 10 subject mice at a time to allow close observation.

      Video 1. A bout of aggression in social defeat by CD-1 aggressor. Approximately 3 to 5 bouts of consecutive aggressive behaviors can be observed during a social defeat period.

    4. For the control group, place C57BL/6J mice pairs in an identical social defeat cage set up with one mouse on each side of perforated Plexiglas divider for the duration of the 10-day social defeat test without any physical contact. The mice are rotated daily between control cages (‘intruder’ side only).
    5. Repeat daily social defeat sessions in a novel CD-1 aggressors’ homecage for 10 days.
    6. Immediately following the last social defeat session, singly house all C57BL/6J intruder mice. Provide animals with ad libitum access to food and water. All CD-1 aggressor mice are returned to their singly-housed cages and used for future social defeat experiments (need to be rescreened).
  3. Social interaction test
    1. Social interaction tests can be performed 24 h after the last social defeat session. In the social interaction test, the CD-1 aggressor mouse should be completely novel to the defeated C57BL/6J mouse being tested. Screen the CD-1 aggressor mouse prior to performing social interaction test to ensure mouse meets criterion for aggression.
    2. Assemble social interaction arenas as shown in Figure 3. Set up video-tracking system (e.g., EthoVision XT) to collect all data automatically by tracking software.


      Figure 3. Schematics of the social interaction test arena. A. Social defeat open field arena and zones for social interaction analysis. B. Removable, perforated Plexiglas enclosure for ‘target’ CD-1 aggressor mice.

    3. Habituate defeated C57BL/6J mice to behavioral testing suite at least 1 h prior to social interaction test (under red light condition, isolated from external noise sources and animals).
    4. Each social interaction test is composed of two 2.5 min phases under red light condition. In the first phase (‘target’ absent), place the C57BL/6J defeated mouse into the social interaction arena (at the center of opposite side to the enclosure) and allow to explore freely without a ‘target’ CD-1 mouse.
      Note: Do not clean the arena after the first phase.
    5. After the first phase, return the defeated C57BL/6J mouse back to its home cage until the second phase. Then, place the ‘target’ CD-1 mouse directly into the removable enclosure.
      Note: Drop the CD-1 mouse gently from top of the enclosure.
    6. In the second phase (‘target’ present), reintroduce the defeated C57BL/6J mouse and let it move freely around in the presence of the ‘target’ CD-1 mouse.
      Note: Place the defeated mice in a consistent manner between phases.
    7. At the end of each test session, remove the target and defeated mouse and place them into their home cage. Sanitize all equipment using cleaning solution.
    8. Data can be analyzed as discussed in Data analysis section.

Data analysis

Using an automated video tracking system, cumulative times in interaction zone and corner zone can be calculated from video data of each trial. Control mice usually spend more time in the interaction zone in ‘target’ present trial, but in defeated mice this kind of social interaction time with the ‘target’ mice is decreased (Figure 4). Thus, social avoidance of mice can be quantified by comparing interaction zone times with or without a ‘target’ mouse (CD-1 aggressor) in the enclosure. Social interaction ratio (SI ratio) is calculated from both interaction zone times as follows:



Mice showing less than 100% of SI ratio are grouped as depression-susceptible mice, and, on the other hand, mice showing over 100% SI ratio belong to depression-resilient group. In most cases, more than half of C57BL/6J mice show susceptible phenotype when they are subjected to chronic social defeat stress. Corner zone time is another useful index of social avoidance, which tends to inversely correlate with the interaction zone time (Figure 4B, lower panel).


Figure 4. Statistic analysis of the social interaction test. A. Representative heatmap of social interaction data for control, susceptible and resilient mice. B and C. Statistical analysis show decreased social interaction with ‘target’ CD-1 mice in susceptible group (n = 18) after social defeat stress, but not in control (n = 23) and resilient groups (n = 13). *P < 0.05, **P < 0.01 vs. ‘no target’, ##P < 0.01 vs. control, ANOVA. Data are represented as the mean ± SEM.

Notes

  1. The cage setup for social defeat stress can be varied, and additional equipment like divider and cylinder can be modified or substituted according to the cage shape and dimension.
  2. Social defeat duration (5-10 min) can be adjusted according to the aggressiveness of CD-1 aggressor mice for appropriate physical stress without severe wounding. Observe each session carefully and replace CD-1 mice that are too aggressive (consistently make open wounds), or not aggressive enough during social defeat sessions. Euthanize severely injured defeated mice.
  3. Variations of social defeat protocol: To shorten the period of stress (e.g., coincidence with viral expression period, such as HSV) or to minimize damage to implanted cannula (e.g., optogenetics), mice can be subjected to shortened social defeat stress paradigms.
    1. Submaximal social defeat stress (Subthreshold or micro-defeat; 1 day protocol): Mice are placed into the CD-1 aggressor’s cage for 5 min, followed by a 15 min break. This defeat session is repeated two more times with novel CD-1 aggressors (Sun et al., 2015; Kim et al., 2016). Pro-susceptibility of drug treatment or gene modulation can be tested with this method, but the protocol has no significant effect on wild-type mice.
    2. Accelerated social defeat stress (4~5 day protocol): Mice are subjected to an accelerated social defeat stress paradigm (two daily defeats for 4~5 days), and the shortened protocol is equally robust in inducing susceptibility as 10-day chronic social defeat protocol (Vialou et al., 2014; Sun et al., 2015; Heller et al., 2016).

Acknowledgments

This work was supported by grants from NIMH and NARSAD (NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation). This protocol is modified from previous social defeat procedures (Berton et al., 2006; Krishnan et al., 2007; Golden et al., 2011).

References

  1. Berton, O., McClung, C. A., Dileone, R. J., Krishnan, V., Renthal, W., Russo, S. J., Graham, D., Tsankova, N. M., Bolanos, C. A., Rios, M., Monteggia, L. M., Self, D. W. and Nestler, E. J. (2006). Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science 311(5762): 864-868.
  2. Golden, S. A., Covington, H. E., 3rd, Berton, O. and Russo, S. J. (2011). A standardized protocol for repeated social defeat stress in mice. Nat Protoc 6(8): 1183-1191.
  3. Heller, E. A., Hamilton, P. J., Burek, D. D., Lombroso, S. I., Pena, C. J., Neve, R. L. and Nestler, E. J. (2016). Targeted epigenetic remodeling of the Cdk5 gene in nucleus accumbens regulates cocaine- and stress-evoked behavior. J Neurosci 36(17): 4690-4697.
  4. Kim, H. D., Hesterman, J., Call, T., Magazu, S., Keeley, E., Armenta, K., Kronman, H., Neve, R. L., Nestler, E. J. and Ferguson, D. (2016). SIRT1 mediates depression-like behaviors in the nucleus accumbens. J Neurosci 36(32): 8441-8452.
  5. Krishnan, V., Han, M. H., Graham, D. L., Berton, O., Renthal, W., Russo, S. J., Laplant, Q., Graham, A., Lutter, M., Lagace, D. C., Ghose, S., Reister, R., Tannous, P., Green, T. A., Neve, R. L., Chakravarty, S., Kumar, A., Eisch, A. J., Self, D. W., Lee, F. S., Tamminga, C. A., Cooper, D. C., Gershenfeld, H. K. and Nestler, E. J. (2007). Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131(2): 391-404.
  6. Krishnan, V. and Nestler, E. J. (2011). Animal models of depression: molecular perspectives. Curr Top Behav Neurosci 7: 121-147.
  7. Russo, S. J. and Nestler, E. J. (2013). The brain reward circuitry in mood disorders. Nat Rev Neurosci 14(9): 609-625.
  8. Sun, H., Damez-Werno, D. M., Scobie, K. N., Shao, N. Y., Dias, C., Rabkin, J., Koo, J. W., Korb, E., Bagot, R. C., Ahn, F. H., Cahill, M. E., Labonte, B., Mouzon, E., Heller, E. A., Cates, H., Golden, S. A., Gleason, K., Russo, S. J., Andrews, S., Neve, R., Kennedy, P. J., Maze, I., Dietz, D. M., Allis, C. D., Turecki, G., Varga-Weisz, P., Tamminga, C., Shen, L. and Nestler, E. J. (2015). ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior. Nat Med 21(10): 1146-1153.
  9. Vialou, V., Bagot, R. C., Cahill, M. E., Ferguson, D., Robison, A. J., Dietz, D. M., Fallon, B., Mazei-Robison, M., Ku, S. M., Harrigan, E., Winstanley, C. A., Joshi, T., Feng, J., Berton, O. and Nestler, E. J. (2014). Prefrontal cortical circuit for depression- and anxiety-related behaviors mediated by cholecystokinin: role of DeltaFosB. J Neurosci 34(11): 3878-3887.

简介

神经精神科研究面临的巨大挑战是准确反映情感障碍症状的动物模型的发展。 已经显示出有效的研究抑郁症的病理学有效模型是慢性社会失败压力模型。 在该模型中,C57BL / 6J小鼠连续10天受CD-1侵袭小鼠诱导的慢性社会失败应激。 这里讨论的是描述CD-1侵略性小鼠的筛选过程的方案,C57BL / 6J和CD-1侵略性小鼠之间的对抗以及社会避免评分的分析作为抑郁症行为的指示。
【背景】抑郁症是全球影响约1.2亿人的复发和危及生命的状况。神经精神科研究领域的重要兴趣是开发有效的抑郁症动物模型,以帮助了解抑郁障碍的神经生物学和分子机制。已经使用各种慢性应激模型来诱导与抑郁有关的小鼠症状,其中包括慢性不可预测的应激,足部休克应激,固定应激,随后测量的是抗缺血或绝望样行为(例如蔗糖偏好测试,强制游泳试验和尾巴悬挂试验),但它们没有被很好地验证为人类抑郁症模型(Krishnan and Nestler,2011)。目前,慢性社会失败压力已经被广泛接受为抑郁症的有效证明模型,因为其可靠性和可重复性(Berton et al。,2006; Krishnan et al。,2007; Golden et al。,2011; Krishnan and Nestler ,2011; Russo和Nestler,2013)。由于以下原因,社会失败模式已被证实为人类抑郁症的良好模式:1)易感小鼠的抑郁症表型(社会避免,慢性反应,代谢变化等)通过慢性治疗抗抑郁药物逆转,不是通过急性管理。 2)社会失败压力产生易感和有弹性的表型,可用于解释抑郁症病理生理学中人类应激易感性的个体差异。 3)由社会失败压力引起的易感表型长期持续,伴随着脑回馈电路的遗传和表观遗传变化。

关键字:社会挫败, 抑郁症, 慢性应激, 社会互动, 小鼠模型

材料和试剂

  1. C57BL/6J(JACKSON LABORATORY):7-8周龄的小鼠被用于社会失败(6-7周小鼠被命令在一个星期适应环境中的生物)
    注意:将小鼠放在标准笼子中,每笼有4至5只小鼠。
  2. CD-1小鼠(Charles River):退休的雄性育种小鼠4-6个月龄,单独饲养在标准小鼠笼中
    注意:社会失败模式使用主体小鼠作为社会压力源的一致从属,并且在本协议中采用最广泛使用的菌株C57BL/6J:CD-1对的强制从属策略。 br />
  3. 清洁溶液(用于清洁竞技场和定制有机玻璃社交互动竞技场/分隔线之间):0.5%过氧化氢

设备

  1. 清洁大鼠笼(带有床上用品的一次性大鼠笼)(Innovive,目录号:RS1-H-C8)
  2. 标准水瓶(小鼠水瓶)(Innovive,目录号:M-WB-300)
  3. 带有吸管的水瓶塞(氯丁橡胶塞,尺寸:#8.5;安心,1"弯管 - 开口)(安检,目录号:OT-199 - 3")
  4. 气瓶(定制,附加喷水器的塑料支架;图2B)
  5. 分隔线(定制,透明有孔玻璃板;图2B)
  6. 可拆卸外壳(定制,透明的有机玻璃,顶部和底部是打开的;图3B)
  7. 秒表
  8. 露天室(定制,44 [w] x 44 [d] x 30 [h] cm)
  9. 用于社交互动测试的视频跟踪系统(例如,,Noldus信息技术,型号:EthoVision系统,CCD相机和计算机)

软件

  1. 跟踪软件:EthoVision XT(Noldus信息技术)

程序

注意:慢性社会失败压力实验必须符合关于使用实验动物的政府和机构准则。

  1. 筛选CD-1侵袭小鼠
    1. 在4-6个月龄订购雄性CD-1退休育种小鼠(订购至少比所需的2-3倍,因为少于一半的CD-1小鼠将具有足够的攻击性)。 CD-1小鼠应单独饲养在标准小鼠笼中,随意获取食物和水。在启动筛选过程之前,让CD-1小鼠适应新环境一周。
    2. C57BL/6J'筛选者'雄性小鼠仅用于筛选CD-1小鼠。筛选小鼠可以在年龄(8-20周)范围内,并可用于随后的筛选过程。去除任何侵略性的"筛选"老鼠(罕见)。
    3. 在宿主CD-1小鼠的笼中进行CD-1小鼠的筛选。 C57BL/6J'筛选器的小鼠应直接放入入侵者CD-1小鼠的家笼中5分钟。应注意每个CD-1的潜伏期。进行三次筛选,连续3天(每天一次),不同的筛选者。
    4. 应根据两个标准选择CD-1侵略性小鼠:在三个5分钟的筛选阶段,初始侵袭的延迟必须在连续两个会话期间小于60秒。并且CD-1必须连续攻击C57BL/6J筛选鼠标至少10秒,而不会中断所有三个5分钟的会话。
    5. 选择用于自由获取食物和水的实验室CD-1侵略鼠(自由使用)(即可使用)。侵略者小鼠可以在初次筛选后使用最多3个月(CD-1侵略性小鼠可以习惯于社会失败,因此需要在下一次社会失败实验之前进行重新筛选)。
  2. 慢性社会失败压力(图1)


    图1.社会失败和社会互动测试示意图。慢性社会失败计划在分开的笼子中使用CD-1攻击性小鼠连续10天的社会失败,随后进行社会互动测试和随后的分析。

    1. 组装社会失败的笼子(图2A),采取标准的鼠笼,并用木片床垫填充笼子底部。将穿孔的有机玻璃隔板放在笼子的中心。分手在失败之后将身体分离小鼠,并允许在非失败住房期间进行感官交互。在标准水瓶侧的相对侧放置圆筒(额外的水瓶架)。插入带有圆柱形支架的组装水瓶。确保水易于接近。将食物放在每边的底部(水和食物应该可以自由获取)。将标准鼠笼的盖子牢固地放在笼子上。 10天的社会失败协议需要至少10个具有CD-1侵略者的社会失败的笼子。
      注意:分隔线应牢固定位,以防止转义。


      图2.慢性社会失败压力下的标准笼设置。 A.用于慢性社会失败压力的标准大鼠笼。常规侵略者CD-1鼠标永久地放置在穿孔分隔器的一侧,而C57BL/6J小鼠在失败后每天旋转,以避免对侵略鼠的习惯。 B.标准鼠笼附加部件尺寸(定制)。气瓶在标准水瓶侧相对的一侧容纳一个额外的水瓶。穿孔的有机玻璃分隔器在非社会失败期间物理分离小鼠(穿孔允许感官交互)。

    2. 在新的笼子中习惯的社会失败会议开始之前,将居民CD-1侵略鼠标放在分隔的家庭笼子的一边。 注意:气缸侧是较小的空间中有效的失败的首选。
    3. 在社会失败会议的第一天,将入侵者C57BL/6J小鼠直接放入CD-1侵略鼠的笼子侧。经过5-10分钟的社交失败(视频1),将入侵者鼠标转移到穿孔分隔线的对面,并在24小时内的剩余时间内放置在车厢内。
      注意:将入侵者鼠标靠近攻击者在会话开始时有助于启动攻击性行为。一次可以与多达10名受试者的小鼠进行社交失败压力,以便密切观察。

      Video 1. A bout of aggression in social defeat by CD-1 aggressor. Approximately 3 to 5 bouts of consecutive aggressive behaviors can be observed during a social defeat period.

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

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    4. 对于对照组,将C57BL/6J小鼠对放置在与穿孔的有机玻璃分隔器两侧的一只小鼠相同的社会失败笼中,持续10天的社会失败测试,没有任何物理接触。控制笼之间每天旋转小鼠(仅限入侵者侧)。
    5. 在CD-1侵略者的小故事中重复每天的社交失败10天。
    6. 在最后一次社交失败之后,单独安置所有C57BL/6J入侵者小鼠。为动物提供免费获取食物和水的动物。所有CD-1攻击性小鼠都被归还到他们单独饲养的笼子中,用于未来的社会失败实验(需要重新筛选)。
  3. 社交互动考试
    1. 社交互动测试可以在最后一次社交失败后24小时进行。在社交互动测试中,CD-1侵略者鼠标对被测试的被击败的C57BL/6J小鼠应该是完全新颖的。在执行社交互动测试之前,先屏蔽CD-1侵略鼠标,以确保鼠标达到侵略的标准。
    2. 组装社交互动领域,如图3所示。设置视频跟踪系统(例如,EthoVision XT)通过跟踪软件自动收集所有数据。


      图3.社交互动测试领域的示意图。 A.社会失败的开放领域竞技场和社区互动分析区域。 B.用于"目标"CD-1侵略鼠的可移动穿孔的有机玻璃外壳。

    3. Habituate在社会互动测试之前至少1小时(在红光条件下,从外部噪声源和动物分离),将C57BL/6J小鼠击败行为测试套件。
    4. 每个社会互动测试由红光条件下的两个2.5分钟相组成。在第一阶段("目标"不存在),将C57BL/6J击败的小鼠放入社交互动场(位于外壳相对侧的中心),并允许自由探索,而无需"目标"CD-1鼠标。 br /> 注意:不要在第一阶段之后清洁竞技场。
    5. 第一阶段后,将被击败的C57BL/6J鼠标返回到其家庭笼,直到第二阶段。然后,将"目标"CD-1鼠标直接放入可移动外壳中。
      注意:从机箱顶部轻轻放下CD-1鼠标。
    6. 在第二阶段("目标")中,重新引入被击败的C57BL/6J鼠标,并在"目标"CD-1鼠标存在的情况下自由移动。
      注意:在相位之间以一致的方式放置被击败的小鼠。
    7. 在每个测试会话结束时,删除目标并击败鼠标并将它们放入家中的笼子中。使用清洁液消毒所有设备。
    8. 可以在数据分析部分中讨论数据进行分析。

数据分析

使用自动视频跟踪系统,可以从每个试验的视频数据计算交互区域和角落区域的累积时间。对照小鼠通常在"目标"现在的试验中在相互作用区域花费更多的时间,但在被击败的小鼠中,这种与"目标"小鼠的社会交互时间减少(图4)。因此,通过与外壳中的"目标"小鼠(CD-1侵略者)或不相关的相互作用区时间进行比较可以量化小鼠的社会避免。社会互动比率(SI比率)由两个交互区时间计算如下:



显示小于100%的SI比的小鼠被分组为抑郁敏感小鼠,另一方面,显示超过100%SI比的小鼠属于抑郁 - 弹性组。在大多数情况下,超过一半的C57BL/6J小鼠在遭受慢性社会失败压力时显示易感表型。角区时间是社会回避的另一个有用的指标,它倾向于与相互作用区时间反向相关(图4B,下图)。


图4.社会互动测试的统计分析。 A.用于控制,易感和有弹性的小鼠的社交交互数据的代表性热图。 B和C.统计分析显示社会失败压力后敏感组(n = 18)与对照组(n = 23)和弹性组(n = 13)之间的"目标"CD-1小鼠的社会互动情况减少。 * 0.05,** P < 0.01 vs.'no target', ## P < 0.01与对照组,方差分析。数据表示为平均值±SEM。

笔记

  1. 用于社会失败应力的笼子设置可以变化,可以根据笼子的形状和尺寸修改或替换分隔件和气缸等附加设备。
  2. 社会失败持续时间(5-10分钟)可以根据CD-1侵略性小鼠的攻击性进行适当的身体压力而不造成严重伤害。仔细观察每个会话,并取代太过激进的(一直打开伤口)的CD-1小鼠,或者在社交失败时不够积极。安乐死严重受伤的失败的老鼠。
  3. 社会失败方案的变化:缩短压力时期(例如,,与病毒表达时期如HSV一致)或减少对植入插管的损害(例如)。 ,光遗传学),小鼠可以经受缩短的社会失败压力范例。
    1. 亚极限社会失败压力(亚阈值或微失败; 1天方案):将小鼠放入CD-1侵略者笼中5分钟,然后15分钟休息。新的CD-1侵略者(Sun等人,2015; Kim等人,2016)再次重复了这次失败的会议。可以用该方法检测药物治疗或基因调控的易感性,但该方案对野生型小鼠无明显影响。
    2. 加速社会失败压力(4〜5天方案):小鼠受到加速的社会失败压力范式(每天2次,每次4〜5天),缩短方案在诱导易感性方面同样强大,为10天慢性社会失败协议(Vialou等人,2014; Sun等人,2015; Heller等人,2016)。

致谢

这项工作得到了NIMH和NARSAD(NARSAD来自脑与行为研究基金会的青年研究者资助)的资助。该协议从以前的社会失败程序(Berton等人,2006; Krishnan等人,2007; Golden等人, 2011)。

参考文献

  1. Berton,O.,McClung,CA,Dileone,RJ,Krishnan,V.,Renthal,W.,Russo,SJ,Graham,D.,Tsankova,NM,Bolanos,CA,Rios,M.,Monteggia,LM,Self ,DW and Nestler,EJ(2006)。  基本角色BDNF在脑膜炎多巴胺途径中的社会失败压力。 科学 311(5762):864-868。
  2. Golden,SA,Covington,HE,3rd,Berton,O. and Russo,SJ(2011)。  小鼠中重复的社会失败压力的标准化协议 Nat Protoc 6(8):1183-1191。
  3. Heller,EA,Hamilton,PJ,Burek,DD,Lombroso,SI,Pena,CJ,Neve,RL和Nestler,EJ(2016)。  伏隔核中的Cdk5基因的靶向表观遗传重建调节可卡因和应激诱发行为。 < (J)Neurosci 36(17):4690-4697。
  4. Kim,HD,Hesterman,J.,Call,T.,Magazu,S.,Keeley,E.,Armenta,K.,Kronman,H.,Neve,RL,Nestler,EJ和Ferguson,D。(2016)。   SIRT1介导伏隔核中的抑郁症行为。/a> J Neurosci 36(32):8441-8452。
  5. Krishnan,V.,Han,MH,Graham,DL,Berton,O.,Renthal,W.,Russo,SJ,Laplant,Q.,Graham,A.,Lutter,M.,Lagace,DC,Ghose, ,Reister,R.,Tannous,P.,Green,TA,Neve,RL,Chakravarty,S.,Kumar,A.,Eisch,AJ,Self,DW,Lee,FS,Tamminga,CA,Cooper,DC,Gershenfeld ,HK and Nestler,EJ(2007)。  分子适应潜在的敏感性和抵抗大脑报酬区域的社会失败。 细胞 131(2):391-404。
  6. Krishnan,V.和Nestler,EJ(2011)。抑郁症的动物模型:分子视角。 Curr Top Behav Neurosci 7:121-147。
  7. Russo,SJ和Nestler,EJ(2013)。情绪障碍中的脑回报电路。 Nat Rev Neurosci 14(9):609-625。
  8. Sun,H.,Damez-Werno,DM,Scobie,KN,Shao,NY,Dias,C.,Rabkin,J.,Koo,JW,Korb,E.,Bagot,RC,Ahn,FH,Cahill,ME, Labonte,B.,Mouzon,E.,Heller,EA,Cates,H.,Golden,SA,Gleason,K.,Russo,SJ,Andrews,S.,Neve,R.,Kennedy,PJ,Maze,I. ,Dietz,DM,Allis,CD,Turecki,G.,Varga-Weisz,P.,Tamminga,C.,Shen,L。和Nestler,EJ(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26390241"target ="_ blank"> ACF染色质重塑复合物介导应激诱导的抑郁样行为。 Nat Med 21(10):1146-1153。
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Copyright: © 2017 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. Kim, H., Call, T., Carotenuto, S., Johnson, R. and Ferguson, D. (2017). Testing Depression in Mice: a Chronic Social Defeat Stress Model. Bio-protocol 7(7): e2203. DOI: 10.21769/BioProtoc.2203.
  2. Kim, H. D., Hesterman, J., Call, T., Magazu, S., Keeley, E., Armenta, K., Kronman, H., Neve, R. L., Nestler, E. J. and Ferguson, D. (2016). SIRT1 mediates depression-like behaviors in the nucleus accumbens. J Neurosci 36(32): 8441-8452.
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