Sensitive Assessment of Hippocampal Learning Using Temporally Dissociated Passive Avoidance Task

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The Journal of Neuroscience
Oct 2015



The temporally dissociated passive avoidance (TDPA) paradigm is a variant of passive avoidance testing, and allows for more sensitive investigation of mild impairments in avoidance learning. Passive avoidance learning measures the latency to enter a “dark” context in which an aversive stimulus (foot shock) has been previously experienced using a light-dark box paradigm. Briefly, the animal is placed into the light side of the box and the time spent to cross into the dark side is measured. After entry into the dark chamber, the animal receives a mild (0.4-1.6 mA) footshock and is removed from the box. After a period of time, typically 24 h (note that this is entirely dependent on whether various levels of memory retention, e.g., short or long, are being measured), the animal is placed back into the box and cross-over latency is measured. Passive avoidance is learned after one trial and results in a robust increase in crossover latency. This behavior requires the association between a normally neutral environment and an aversive stimulus, and is dependent on hippocampal function (Stubley-Weatherly et al., 1996; Impey et al., 1998). TDPA extends this learning across multiple once-daily trials, producing a more graded and malleable latency score, and thus allows a more sensitive evaluation of changes in hippocampal function The task remains dependent on an intact hippocampus (Zhang et al., 2008), and subtle changes in hippocampal gene expression can result in robust alterations in TDPA latency scores (Eagle et al., 2015). We describe here a common method used to assess TDPA learning in mice.

Keywords: Memory (记忆), Fear (恐惧), Hippocampus (海马), Mouse (鼠标), Behavior (行为)

Materials and Reagents

  1. Adult (7 weeks or older) mice (C57BL/6J) (the Jackson laboratory)
    Note: C57BL/6J mice are typical, though alternate strains and ages of mice may also be used. Mice are housed singly or in groups of 4-5 per cage. Conditions should comply with the Guide for the Care and Use of Laboratory Animals, 8th ed. ( See Animals considerations in Notes for more details.
  2. Ethanol (70% volume/volume) diluted in distilled water


  1. Light-dark box (14 in. W x 7 in. D x 12 in. H) equipped with overhead houselight (light side only), guillotine-style door, drop pan, and shock-capable grid flooring (Coulbourn Instruments, model: H10-11M-PA )
  2. Precision animal shocker (Coulbourn Instruments, model: H13-15 ) and cable
  3. Video camera, IR-capable (Panasonic Corporation of North America, model: WV-CP304 ), mounted in front of box with side-view of light chamber and door
  4. Timer
  5. Sound-attenuating cubicle, as needed
    Note: For example, Coulbourn Instruments provides a range of isolation cubicles in different sizes (H10-24 series) that are suitable for the needs of this experiment.


  1. Set up equipment (Figure 1). Clean the light-dark chamber with 70% ethanol/water solution on all surface areas and wipe clean. Make sure doorway between light side and dark side is closed. Turn on houselight. Turn on shocker and adjust for pre-determined shock intensity (see below). 

    Figure 1. Example of a light-dark box

  2. Place mouse in light chamber of the light-dark box. Wait approximately 60 sec for mouse to acclimate to light side. Mice will freely explore the entire light side. Before opening the door, ensure the mouse is facing away from the door (at least 45°) and at least 5 cm from the door. This prevents impulsive entry upon door movement. Note that the door will make an audible click upon opening.
  3. Raise the door separating the dark side from the light side of the light-dark box. Start the timer to measure cross-over latency, defined as the duration after door opens for mouse to enter the dark side of the light-dark box. Crossover is defined by full body entry (including tail). Stop the timer when the mouse enters the dark side of the chamber.
    Note: Mice may enter halfway and dart back into the light side. This is more likely after repeated daily trials. Do not lower the door until the mouse has fully crossed over.
  4. Once the mouse has crossed over, lower the door. After repeated trials, most non-experimental mice (e.g., wild-type C57BL/6J with no manipulation) will reach criterion. Criterion typically consists of 300-600 sec in the light side without any crossover. The determination of the criterion is entirely dependent on the experiment. Once an animal has reached criterion it is manually removed from the light side (no footshock is given). It no longer undergoes additional testing and crossover latency is considered maximal for remaining sessions.
  5. After a specific amount of time in the dark side (see below), apply a single foot-shock to the grid floor. The amount of time and the shock intensity will determine (affect) cross-over latency in subsequent trials (see Figure 2).

    Figure 2. Hypothetical effects of varying delay to shock and shock intensity in the TDPA task. A. Increases in delay to shock (2 sec-10 min) reduce latency to crossover. B. Increases in shock intensity (0.0-1.6 mA) increase latency to crossover.

  6. Allow the mouse to remain in the dark chamber for 30 sec after the foot shock to associate the environment with the aversive stimulus, then remove mouse to home cage.
  7. All the urine and fecal boli must be removed from both sides of the chamber, and the chamber cleaned entirely with 70% ethanol to remove any residual smell from the first mouse. Afterwards, the next mouse may be submitted to the test.
  8. Repeat procedure until all mice have been tested.
  9. Repeat procedure each day for 5 daily sessions.

    Video 1. Demonstration of temporally dissociated passive avoidance procedure in mice.

    Note: This video was recorded with the room lighets on. Normally, room lights are turn off.


  1. Determining timing and intensity of shock
    Increasing the time between entry into the dark chamber and foot shock and/or decreasing shock intensity can be used to achieve greater sensitivity for detection of mild learning impairments (Figure 2). For instance, if a robust reduction in hippocampal function resulting in an expected increase in crossover latency is hypothesized, a strong shock (1.2 mA) and/or short time before shock (30 sec) could be used to elicit maximal differences between groups. In contrast, if a mild reduction in hippocampal function is hypothesized, a moderate shock (0.4-0.8 mA) and time before shock (5-10 min) could be used to detect a modest reduction in crossover latency, particularly in later sessions. Across five days of TDPA, mild impairment in hippocampal function results in a rightward shift in the learning curve (Eagle et al., 2015). However, it should be expected that increasing time and decreasing intensity will also contribute to greater between-subject variability and across-trial variability. For extended (e.g., 5-day) learning, times ranging from 30 sec to 600 sec and intensity ranging between 0.4-1.2 mA are recommended. Other variables may also alter behavior, including time to criterion. While we provide suggested parameters, we strongly recommend conducting preliminary studies to address the needs of your own experiments.
  2. Measurement/control
    The protocol we present is based on a manual setup. However, many hardware and software suppliers allow for alternative methods of measurement and testing control. Measurements can be achieved by automated video tracking or photobeam systems. Typical measurements consist of crossover latency across trials (Figure 2) and survival graph depicting the % of animals (per experimental condition) reaching criterion across trials. No behavior is recorded during the shock because the mouse is in an opaque dark chamber. Crossover latency to enter the dark side (from the light side) is the only behavior that is assessed.
  3. Environment and other factors
    It is critical to control all aspects of the environment in order to achieve consistent, replicable results. Testing should always occur in a controlled environment with minimal extraneous sounds or visual and olfactory stimuli. This is best achieved by conducting testing in a quiet, dim room and may be further enhanced by placing the light-dark box in a sound attenuating cubicle. As circadian condition has been shown to affect learning and memory, we recommend that training and testing be performed with fixed time span for all sessions (e.g., 2-4 h into light cycle to determine behavior in the inactive phase or 2-4 h into dark cycle for the active phase).
  4. Animal considerations
    It is important to conduct preliminary studies in order to characterize the behavioral phenotype with particular equipment, environment, and animals. This is especially true in the case of varying mouse strains, either inbred or outbred, if mice have been surgically or behaviorally manipulated prior to TDPA testing, or for determining differences in juvenile mice or sex effects. If factors such as genetic or pharmacological alteration are involved, it is important to determine whether these factors affect sensitivity to foot shock and spontaneous behavior in the light-dark box. We recommend first running a 5-10 min light-dark box test to examine the number of transitions between the two chambers and the total time spent in the light chamber. In addition, some labs prefer to pre-handle mice for 5-7 days and singly house the animals prior to TDPA training/testing.


This behavioral procedure was adapted from previously published studies (Wang et al., 2004; Zhang et al., 2008; Wang et al., 2009) and was performed by our group as described (Eagle et al., 2015). This work was supported by the Whitehall Foundation (AJR; 2013-08-43), the Multidisciplinary Training in Environmental Toxicology training grant (ALE; T32-ES007255), a 2014 NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (ALE), and NIH (HW, MH093445).


  1. Eagle, A. L., Gajewski, P. A., Yang, M., Kechner, M. E., Al Masraf, B. S., Kennedy, P. J., Wang, H., Mazei-Robison, M. S. and Robison, A. J. (2015). Experience-dependent induction of hippocampal DeltaFosB controls learning. J Neurosci 35(40): 13773-13783.
  2. Impey, S., Smith, D. M., Obrietan, K., Donahue, R., Wade, C. and Storm, D. R. (1998). Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning. Nat Neurosci 1(7): 595-601.
  3. Institute of Laboratory Animal Resources (U.S.) (2011). Guide for the care and use of laboratory animals, 8th edition. Washington DC: National Academy of Sciences.
  4. Stubley-Weatherly, L., Harding, J. W. and Wright, J. W. (1996). Effects of discrete kainic acid-induced hippocampal lesions on spatial and contextual learning and memory in rats. Brain Res 716(1-2): 29-38.
  5. Wang, B., Hu, Q., Hearn, M. G., Shimizu, K., Ware, C. B., Liggitt, D. H., Jin, L. W., Cool, B. H., Storm, D. R. and Martin, G. M. (2004). Isoform-specific knockout of FE65 leads to impaired learning and memory. J Neurosci Res 75(1): 12-24.
  6. Wang, Y., Zhang, M., Moon, C., Hu, Q., Wang, B., Martin, G., Sun, Z. and Wang, H. (2009). The APP-interacting protein FE65 is required for hippocampus-dependent learning and long-term potentiation. Learn Mem 16(9): 537-544.
  7. Zhang, M., Moon, C., Chan, G. C., Yang, L., Zheng, F., Conti, A. C., Muglia, L., Muglia, L. J., Storm, D. R. and Wang, H. (2008). Ca-stimulated type 8 adenylyl cyclase is required for rapid acquisition of novel spatial information and for working/episodic-like memory. J Neurosci 28(18): 4736-4744.


时间分离的被动回避(TDPA)模式是被动回避测试的一个变体,允许对避免学习中的轻度损伤进行更敏感的调查。被动回避学习衡量潜伏期进入“黑暗”环境,在此背景下,使用暗黑盒子模式曾经使用过厌恶刺激(足震)。简而言之,将动物放置在盒子的光侧,并测量穿过黑暗面的时间。进入暗室后,动物接受轻度(0.4-1.6毫安)的脚蹬,并从箱子中取出。经过一段时间,通常为24小时(注意,这完全取决于是否测量各种记忆保留水平,例如短时间或长时间),将动物放回盒子中,并测量交叉潜伏期。被动避免在一次试用后被学习,并导致交叉延迟强劲增加。这种行为需要通常中性环境和厌恶刺激之间的关联,并且依赖于海马功能(Stubley-Weatherly等人,1996; Impey等人,1998)。 TDPA通过多次每日一次的试验来扩展这种学习,产生更分级和可延展的延迟评分,从而允许对海马功能变化进行更敏感的评估任务依赖于完整的海马(Zhang et al。,2008)和海马基因表达的微妙变化可导致TDPA潜伏期评分的强烈改变(Eagle et al。,2015)。我们在这里描述一种常用的评估小鼠TDPA学习的方法。

关键字:记忆, 恐惧, 海马, 鼠标, 行为


  1. 成年(7周龄或以上)小鼠(C57BL/6J)(Jackson实验室)
    注意:C57BL/6J小鼠是典型的,尽管也可以使用小鼠的替代品系和年龄。 小鼠单独圈养或每笼4-5只。 条件应符合"实验动物护理和使用指南",第8版。 ( )。 有关详情,请参阅Notes中的动物注意事项。
  2. 用蒸馏水稀释的乙醇(70%体积/体积)


  1. 配备有架空室内灯(仅有光侧),闸门型门,滴盘和可冲击的网格地板(Coulbourn Instruments,型号:C-1)的浅色暗盒(14英寸×7英寸×12英寸H) H10-11M-PA)
  2. 精密动物冲击器(Coulbourn Instruments,型号:H13-15)和电缆
  3. 摄像机,支持IR(Panasonic Corporation of North America,型号:WV-CP304),安装在盒子前面,具有光室和门的侧视图
  4. 计时器
  5. 消声柜,根据需要
    注意:例如,Coulbourn Instruments提供了一系列适合本实验需要的不同尺寸(H10-24系列)的隔离柜。


  1. 设置设备(图1)。在所有表面区域用70%乙醇/水溶液清洁光暗室,并擦拭干净。确保明亮和暗侧之间的门口关闭。打开室内照明。打开防震器,调整预先确定的防震强度(见下文)。


  2. 将鼠标放在光暗箱的光室中。等待约60秒,使鼠标适应光线。小鼠将自由探索整个光线。在打开门之前,确保鼠标朝向离开门(至少45°),并且距离门至少5 cm。这防止门移动时的冲击性进入。注意,打开时门会发出一个可听到的咔嗒声。
  3. 抬起门,把暗黑暗的一面与明暗箱的光明一面分开。启动计时器以测量交叉延迟,定义为门打开后鼠标进入亮暗箱暗侧的持续时间。交叉由完整身体条目(包括尾部)定义。当鼠标进入暗室的暗侧时停止计时器。
  4. 一旦鼠标已经越过,放下门。在重复试验后,大多数非实验小鼠(例如,未操作的野生型C57BL/6J)将达到标准。标准通常包括在没有任何交叉的光侧的300-600秒。标准的确定完全取决于实验。一旦动物达到标准,它从轻的一侧被手动移除(没有给出脚步)。它不再经历额外的测试,交叉延迟被认为是剩余会话的最大
  5. 在黑暗面(见下文)中经过特定时间后,将单次足部电击施加到网格地板。时间量和冲击强度将在随后的试验中确定(影响)交叉延迟(参见图2)。

    图2. TDPA任务中不同延迟对休克和休克强度的假设影响。 A.延迟休克延迟(2秒 - 10分钟)减少延迟到交叉。 B.冲击强度的增加(0.0-1.6mA)增加交叉的延迟。

  6. 让鼠标在足底休克后保持在暗室中30秒,使环境与厌恶刺激相关联,然后将鼠标移至笼子。
  7. 所有尿液和粪便boli必须从腔室的两侧移除,并且腔室用70%乙醇完全清洁以从第一只小鼠除去任何残余气味。之后,下一个鼠标可能会提交测试。
  8. 重复程序,直到所有小鼠都经过测试
  9. 每天重复步骤5次。

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  1. 确定冲击的时机和强度
  2. 测量/控制
  3. 环境和其他因素
  4. 动物考虑
    重要的是进行初步研究,以特定的行为表型与特定的设备,环境和动物。如果在TDPA测试之前已经通过手术或行为操纵小鼠,或者用于确定幼年小鼠的差异或性效应,则在不同的小鼠品系(近交系或远交系)的情况下尤其如此。如果涉及诸如遗传或药理学改变的因素,重要的是确定这些因素是否影响对脚部休克和自发行为在亮暗箱中的敏感性。我们建议首先运行5-10分钟的暗 - 暗盒测试,以检查两个腔室之间的过渡的数量和在光腔中花费的总时间。此外,一些实验室喜欢预处理小鼠5-7天,在TDPA训练/测试之前单独安置动物。


该行为过程改编自先前公开的研究(Wang等人,2004; Zhang等人,2008; Wang等人, 2009),并且由我们的小组进行(Eagle等人,2015)。这项工作得到白厅基金会(AJR; 2013-08-43),环境毒理学多学科培训(ALE; T32-ES007255),2014年NARSAD年轻研究员奖从大脑和行为研究基金会(ALE) ,和NIH(HW,MH093445)。


  1. Eagle,A.L.,Gajewski,P.A.,Yang,M.,Kechner,M.E.,Al Masraf,B.S.,Kennedy,P.J.,Wang,H.,Mazei-Robison,M.S.and Robison,A.J。 经验依赖性诱导海马DeltaFosB对照学习 J Neurosci < em> 35(40):13773-13783。
  2. Impey,S.,Smith,D.M.,Obrietan,K.,Donahue,R.,Wade,C.and Storm,D.R。(1998)。 在上下文学习期间刺激cAMP反应元件(CRE)介导的转录。 Nat Neurosci 1(7):595-601。
  3. 实验动物资源研究所(美国)(2011)。  实验动物护理和使用指南,第8版华盛顿特区:美国科学院。
  4. Stubley-Weatherly,L.,Harding,J.W.and Wright,J.W。(1996)。 离散的红藻氨酸诱导的海马病变对大鼠的空间和语境学习和记忆的影响。 a> Brain Res 716(1-2):29-38。
  5. Wang,B.,Hu,Q.,Hearn,M.G.,Shimizu,K.,Ware,C.B.,Liggitt,D.H.,Jin,L.W.,Cool,B.H.,Storm,D.R.and Martin,G.M。 亚型特异性敲除FE65导致受损的学习和记忆。 J Neurosci Res 75(1):12-24。
  6. Wang,Y.,Zhang,M.,Moon,C.,Hu,Q.,Wang,B.,Martin,G.,Sun,Z.and Wang,H。 APP相互作用蛋白FE65是海马依赖性学习和长期增强所必需的。 a> Learn Mem 16(9):537-544。
  7. Zhang,M.,Moon,C.,Chan,G.C.,Yang,L.,Zheng,F.,Conti,A.C。,Muglia,L.,Muglia,L.J.,Storm,D.R.and Wang, Ca刺激的8型腺苷酸环化酶是快速获取新型空间信息和工作/发作所需的like memory。 J Neurosci 28(18):4736-4744。

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Copyright: © 2016 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. Eagle, A. L., Wang, H. and Robison, A. J. (2016). Sensitive Assessment of Hippocampal Learning Using Temporally Dissociated Passive Avoidance Task. Bio-protocol 6(11): e1821. DOI: 10.21769/BioProtoc.1821.
  2. Eagle, A. L., Gajewski, P. A., Yang, M., Kechner, M. E., Al Masraf, B. S., Kennedy, P. J., Wang, H., Mazei-Robison, M. S. and Robison, A. J. (2015). Experience-dependent induction of hippocampal DeltaFosB controls learning. J Neurosci 35(40): 13773-13783.