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The Object Context-place-location Paradigm for Testing Spatial Memory in Mice

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Jun 2016



This protocol was originally designed to examine long-term spatial memory in PKMζ knockout (i.e., PKMζ-null) mice (Tsokas et al., 2016). Our main goal was to test whether the ability of these animals to maintain previously acquired spatial information was sensitive to the type and complexity of the spatial information that needs to be remembered. Accordingly, we modified and combined into a single protocol, three novelty-preference tests, specifically the object-in-context, object-in-place and object-in-location tests, adapted from previous studies in rodents (Mumby et al., 2002; Langston and Wood, 2010; Barker and Warburton, 2011). During the training (learning) phase of the procedure, mice are repeatedly exposed to three different environments in which they learn the spatial arrangement of an environment-specific set of non-identical objects. After this learning phase is completed, each mouse receives three different memory tests configured as environment mismatches, in which the previously learned objects-in-space configurations have been modified from the original training situation. The mismatch tests differ in their cognitive demands due to the type of spatial association that is manipulated, specifically evaluating memory for object-context and object-place associations. During each memory test, the time differential spent exploring the novel (misplaced) and familiar objects is computed as an index of novelty discrimination. This index is the behavioral measure of memory recall of the previously acquired spatial associations.

Keywords: Space (空间), Novelty preference (新奇偏好), Discrimination memory (歧视记忆), Recognition memory (识别记忆), Context (环境)


The behavioral neuroscientist’s toolkit of laboratory rodent behaviors has needed to expand to both propel and keep up with the progress of the program to identify the molecular mechanisms of memory. The traditional approach has been to use reinforced behaviors to control learning and formation of memory and limit behavior to readily quantified endpoint measures. However, to meet the growing sophistication of the learning and memory field and to test the generality of the postulated mechanisms, the toolkit has needed to expand to multiple estimates of memory. It has been especially important to use memory assays that minimize the need for behavioral shaping (a form of learning itself) and the manipulation of other motivational factors, many of which potentially elicit stress and other potential confounds of the molecular basis of memory (Lesburguères et al., 2016).

Unlike behavioral paradigms that rely on conditioned responses, novelty-preference tasks exploit the rodent’s spontaneous exploratory behavior and its innate tendency to investigate instances of change (novelty) in a familiar environment (Steckler et al., 1998a). The basic reasoning is that memory for the familiar condition is estimated by the extent to which behavior differs in response to novelty. Accordingly, the fundamental principle of a novelty-preference memory paradigm is to experimentally create a ‘non-matching’ condition between the learning (or encoding) phase and the memory test, such that the animal will express its memory of the original learning experience by preferentially exploring the novel stimuli over the familiar ones.

Novelty-preference tasks are easy-to-use and offer a great versatility for investigating cognitive functions such as spatial memory in rodents (Kinnavane et al., 2015). The following protocol is inspired by previous studies that have used different spatial versions of the novelty-preference paradigm, specifically the object-in-place, object-in-context and object-location task variants (Mumby et al., 2002; Langston and Wood, 2010; Barker and Warburton, 2011). Typically, these task protocols consist of a short learning phase followed by a memory retention test, during which the original spatial configuration of the environment is modified in a specific way. In prior studies however, the different task variants have been presented separately, requiring independent groups of animals to be tested. In addition, the tests are often at short delays (minutes to hours) rather than days, as required to specifically evaluate mechanisms of long-term memory persistence. In the present experimental design each memory retention test is performed to evaluate memory lasting at least 24 h in the same animal and corresponds to a specific spatial manipulation of the original learning experience. Additionally, by varying the number of objects that are presented during the learning phase (i.e., 4 objects versus 2 objects), the following protocol allows direct manipulation of the amount of information to be remembered, not only the type of information.

Here we present the behavioral protocol that was recently used to assess the molecular mechanisms of 24-h long-term memory persistence using wild-type and PKMζ knockout mice (Tsokas et al., 2016). The protocol was sensitive enough to reveal that the molecular mechanisms that are crucial for object-in-place associations amongst four objects require the persistent kinase PKMζ, whereas non-PKMζ dependent mechanisms are sufficient for the maintenance of object-in-context associations and object-location associations involving two objects. A similar dissociation between the neural mechanisms that support object-in-place and object-in-context associations is also observed at the level of the dorsal hippocampus. In particular, permanent or temporary lesion of dorsal hippocampus is sufficient to impair acquisition of object-in-place associations but not object-in-context associations (Langston and Wood, 2010; reviewed by Langston et al., 2010), with a potentially greater role of the dentate gyrus-CA3 subcircuit (Lee et al., 2005). Object-in-context associations seem to critically depend on postrhinal cortex function (Norman and Eacott, 2005) whereas object-location associations tend to be sensitive to hippocampus manipulations (Hardt et al., 2010; Barker and Warburton, 2011). Although these dissociations suggest sharp dependencies of the cognitive function each test evaluates on distinct brain regions, the boundaries of these structure-function associations may not be definitive, because the consequences of the lesions depend on task parameters, including whether contextual cues are local or distal and whether the role of a structure is being evaluated by tests of memory acquisition and consolidation or tests of memory retention (reviewed by Langston et al., 2010). Indeed, recognition memory, a subset of which is assessed by the object context-place-location paradigm that we here describe, appears to be mediated by two extended networks of structures one including the hippocampus that is specialized for spatial recognition memory and the other including postrhinal cortex that is specialized for non-spatial/item recognition memory (reviewed by Steckler et al., 1998b).

Materials and Reagents

  1. 3 open boxes (31 x 31 x 19 cm, L x W x H). They are large, solid-bottom, polysulfone cages purchased from Thoren Caging Systems (Hazelton, PA)
  2. 3 different sets of laminate sheets displaying patterns of black shapes on white background (see Figure 1).
  3. 10 non-identical objects
    Note: We used a combination of plastic toys (PetCo®, USA), flasks and jars that differ in shape and/or materials that are difficult to chew, such as strong natural rubber, Pyrex®, polypropylene, and aluminum. These are the objects that the mice will explore. Each object was unique but had approximately equal size, and they were tall enough to prevent the mice from climbing on the objects. The footprint dimension was approximately 6 cm and the height was 17 cm. The objects must be removable and should be easily washed (see Figure 1)
  4. Wild-type male adult (4-month old) mice (C57BL6/J, THE JACKSON LABORATORY)
    1. In the original publication of this protocol, we have also used adult mutant male mice from the PKMζ knockout mouse line, (PKMζ-null, originally described in Lee et al., 2013) which were bred from breeders provided by Robert O. Messing (University of Texas, Austin, TX).
    2. Mice were housed individually, in an environment with controlled temperature (23 °C) and humidity, under a 12-12 h light-dark cycle with ad libitum access to food and water. All behavioral procedures were conducted during the 7 AM-7 PM light phase of the cycle. While it was not tested, training and testing with a reverse light cycle should have no impact on the behavioral outcomes, as long as the housing and training conditions are consistent. Mice were housed individually in dual cages (2 individual compartments per cage; 1 Wild Type and 1 Knockout per cage). The mutant animals were bred in our facility, and the genotype is known when the animals are weaned. Other mice in the study were housed individually because they were implanted with intracerebral injection cannulae or microelectrode arrays that can be damaged by another mouse. Consequently, we opted for individual housing to ensure similar housing conditions between all animals. Alternative housing, such as in pairs, might be preferable when practical.
  5. Water and 70% EtOH


  1. Apparatus
    The experiments are conducted in the 3 open plastic boxes, customized with different patterns in order to make 3 distinct contexts (A, B and C). For each context, 3 of the 4 inside walls are covered by laminate paper sheets displaying a specific pattern with a strong contrast so that the contexts can be easy to discriminate for the mice. We created walls with a repeating pattern of a black shape on white background (either stripes or dots, see Figure 1) for two of the contexts, and used an all white laminate for the third context. The fourth wall is always left transparent and in the south position, to provide an orientation cue in each context. Each box is placed at the center of the experimental room on an elevated support.

    Figure 1. Example of the three different training contexts A, B and C with their specific set of objects. Upper panel: three of four walls are covered with a specific visual pattern. The fourth wall at the south is always left clear. Lower panel: the objects are non-identical and each context contains a unique set of objects. Note that the bright lighting as captured in these pictures is only for illustration, lighting should be dim, to be optimally comfortable for the mice and allow them to see and discriminate the objects. A 10-15 lux light intensity is recommended.

  2. Objects
    The objects (4 objects/context for A and B, 2 objects for context C) are fixed on the floor with removable adhesive putty such that their edges are 5 cm away from the walls. The precise position of each object is always the same (see Notes for additional comments). To ensure that the objects are repositioned in the same configuration after cleaning between trials, we recommend marking the position on the bottom of both the objects and the box.
  3. Overhead camera (Firefly USB 2.0 camera, FLIR Integrated Imaging Solutions, catalog number: FMVU-03MTM-CS ), equipped with ½” 4-12 mm CCTV Lens (TAMRON, catalog number: 12VM412ASIR ) and software (Tracker, Bio-Signal Group, Acton, MA) for digital video tracking of the mouse’s position and recording the overall behavior on video
  4. Tracking system
    We used a PC-controlled video tracking system (Tracker, Bio-Signal Group, Acton, MA) to accurately detect the position of the animal in each context during the behavioral sessions and to record its exploratory behavior for offline analysis. The position of the mouse was taken to be the centroid of its image in each 1/30 sec video frame.


  1. One week prior to the behavioral experiments, each day, all animals are familiarized to the transportation procedure from the housing location to the experimental room. The animals are also handled in the experimental room by the experimenter to habituate them to the procedure.
  2. Each day, mice are placed in the experimental room 30 min before the beginning of the behavioral experiments for acclimation.
  3. The duration of the entire behavioral procedure is about 9 consecutive days. As depicted below in Figure 2, each mouse is trained and tested in the object-context-place tasks first, followed by the object-location task. Each task procedure consists of three phases: pre-training, training and retention test.

    Figure 2. Experimental design of the object context-place-location paradigm

    1. Day 1: Pretraining in contexts A and B
      1. Animals are habituated to contexts A and B. They explore each box for 10 min with no objects present. Each pretraining session is separated by a 1-h inter-trial interval. The pretraining session starts immediately after the animal has been placed, by hand, at the center of the box, its nose facing the south wall.
      2. Between each pretraining session, the boxes and objects are cleaned with water followed by 70% EtOH, which is allowed to dry. This is done to prevent build up of olfactory cues.
      3. At the end of the 10 min of exploration the animal is removed from the context and returned to its home cage.
      Note: The order of context exposures is counterbalanced between animals within each experimental group, which was genotype in the case of Tsokas et al. (2016). 
    2. From Day 2 to Day 4: Training in contexts A and B
      The mice are allowed to explore contexts A and B, each during two 5-min trials/day, separated by a 1-h inter-trial interval. These trials allow the mice to learn the spatial arrangement of the four objects that are associated with each context.
      Note: The order of each context exploration is counterbalanced between training days and between animals within each experimental group (Table 1).

      Table 1. Example of counterbalancing the order of context exploration (i.e., Contexts A vs. B) between training days and the order within each experimental group (i.e., Wild Type vs. PKMζ-null in the original study)

    3. Day 5: Retention test #1
      Mice are given a first memory retention test that is either an object/context or an object/place mismatch test. In the object/context mismatch test, two of the four objects that had only been encountered in one context are placed in the second context, whereas in the object/place mismatch test two objects from one four-object configuration are place-permuted in the same context they had previously been encountered. The animal is allowed to explore for 3 min. The 3-min duration of the retention test session has been carefully validated. After 3 min the mice become familiar with the novel object configuration and so spatial novelty triggered by the object permutation is no longer detected by the animal. The mice will subsequently explore all the objects equally, whether or not there is a mismatch.
    4. Day 5: Re-exposure
      The same day, an hour after the memory retention test, the mice receive an additional training session (5-min exploration twice in each context A and B) in order to minimize the potential effects of learning the mismatch object-space associations that might have been induced by the retention test.
    5. Day 6: Retention test #2
      Mice are given a second 3-min memory retention test, either an object/context or an object/place mismatch test, whichever test was not administered on Day 5.
      Note: The order of the retention tests on Days 5 and 6, the objects and the places that are permuted, are counterbalanced between animals within each experimental group (Figure 3).

      Figure 3. Counterbalancing Object and/or Place permutations and Retention Test order. A. Examples of spatial-object counterbalancing by making object pair permutations in the Object/Place and Object/Context mismatch tests. Because each subject can have an idiosyncratic preference or dislike for a particular object and/or context, it is important to counterbalance the object-context presentations amongst the subjects within each group during memory tests. There are a very large number of permutations possible. Consider randomizing the objects within a context by exchanging pairs of objects along the horizontal, vertical and two diagonals to generate six unique patterns of permutations and exchanging half of the objects from each context, which will generate 12 object-context arrangements. The six examples of objects permutations represented above from the initial training configuration (the training configuration is displayed as a-b-c-d) in Context A are b-a-c-d (horizontal), a-d-c-b (vertical), a-c-b-d (diagonal); they illustrate counterbalancing of the spatial manipulations for the Object/Place mismatch test. Configurations e-f-c-d (horizontal), a-f-c-h (vertical), a-f-g-d (diagonal) illustrate examples of counterbalancing spatial manipulations for the Object/Context mismatch test. B. Example of counterbalancing the order of the retention tests on Days 5 and 6 (i.e., Object/Place vs. Object/Context mismatch tests) and the type of object permutations between animals within each experimental group (e.g., Wild Type vs. PKMζ-null).

    6. Day 7: Pretraining in context C
      Mice are habituated to explore a new context, context C, for 10 min.
    7. Day 8: Training in context C
      Mice are allowed to explore context C for three sessions of five minutes separated by 1-h inter-trial interval, to learn the spatial configuration of two new objects in a novel environment.
    8. Day 9: Retention test #3
      Mice are given a 3-min retention trial consisting of an object/location mismatch test, in which the location of one object is changed.
      Note: The relocated object and its relative relocation in the context are also counterbalanced between animals within each experimental group.
    9. Behavioral measures of memory performance
      Each video is analyzed offline to manually score the time the mouse is engaged in exploration of an object for each of the retention tests. Object exploration is defined as the nose of the animal being oriented toward the object at a distance of < 2 cm. Each video is therefore replayed in the Tracker software using a 2 cm wide annular mask around each object to define the object exploratory area. Within this area, animal’s activity such as sniffing or touching the object with paws is counted as object exploratory activity only if the animal’s nose is orientated toward the object (see Notes). Measuring object exploration is performed by an experimenter who is blind to the animal’s experimental group and whether the objects have been changed. Memory performance in the three different memory tests are quantified and analyzed using a discrimination index calculated as the absolute difference in time spent exploring the changed (i.e., incorrect, misplaced or relocated) objects and the unchanged objects divided by the total time spent exploring all the objects. As such, the index takes into account individual differences in the total amount of exploration. Good memory retention corresponds to a positive discrimination index, which reflects that the animal was spending more time exploring the incorrect (object/context mismatch), displaced (object/place mismatch) or relocated (object/location mismatch) objects than the objects that had remained unchanged (Figure 4).

      Figure 4. Representative data of behavioral performance. Data can be represented as separate dot plots for each test, depicting the distribution of individual memory performance within each group (e.g., Wild type vs. PKMζ-null). Black bars: Mean ± SEM. The graph is adapted from the data in Tsokas et al., 2016.

Data analysis

Memory performance is analyzed using a one-way ANOVA with repeated measures. The individual effects of the Independent Factor (Group) and the Within-Subjects Factor (Retention test), as well as the Interaction and post-hoc tests are considered significant at an alpha level of 0.05.


  1. To make each to-be-explored object unique and identifiable, we recommend using a variety of shapes and type of materials. The choice of the set of objects should be validated prior to the experiment by testing each configuration set with mice in a pilot study to avoid obviously biased preference to investigate one object over the others.
  2. Animals should not be able to climb the objects, because it would affect the measurement accuracy of the exploratory activity.
  3. The lighting of the objects should be homogenous to avoid creating shadows at the corners of the experimental box. Dark corners are typically preferred by mice and the object-biased presence of preferred places could therefore induce a place preference that could interfere with the overall exploratory activity of the animals.
  4. A quiet, dimly lit (10-15 lux) experimental room is the preferred environment for spontaneous exploratory behavior in mice.
  5. At the end of each day of the behavioral experiment, animals are all left undisturbed for one hour before they are transported back to the animal housing facility.
  6. All behavioral sessions are recorded using the video-tracking system. Whereas the object exploratory activity is a behavioral measure that could only be assessed with the objects present in a given context (during training and memory retention tests), general locomotor activity measures and animal tracking are performed during the pretraining session on Day 1 to assess group differences in locomotion and general exploratory activity, differences of which can bias assessment of novelty preference on the retention tests.


This behavioral protocol was originally used in Tsokas et al., 2016. This work was supported by NIH grants R21NS091830 and R01MH099128 (AAF) and R37MH057068, R01MH53576, R01DA034970, and the Lightfighter Trust (TCS).


  1. Barker, G. R. I. and Warburton, E. C. (2011). When is the hippocampus involved in recognition memory? J Neurosci 31(29): 10721-10731.
  2. Hardt, O., Migues, P. V., Hastings, M., Wong, J. and Nader, K. (2010). PKMζ maintains 1‐day‐and 6‐day‐old long‐term object location but not object identity memory in dorsal hippocampus. Hippocampus 20(6): 691-695.
  3. Kinnavane, L., Albasser, M. M. and Aggleton, J. P. (2015). Advances in the behavioural testing and network imaging of rodent recognition memory. Behav Brain Res 285: 67-78.
  4. Langston, R. F., Stevenson, C. H., Wilson, C. L., Saunders, I. and Wood, E. R. (2010). The role of hippocampal subregions in memory for stimulus associations. Behav Brain Res 215:275-291.
  5. Langston, R. F. and Wood, E. R. (2010). Associative recognition and the hippocampus: differential effects of hippocampal lesions on object-place, object-context and object-place-context memory. Hippocampus 20(10): 1139-1153.
  6. Lee, A. M., Kanter, B. R., Wang, D., Lim, J. P., Zou, M. E., Qiu, C., McMahon, T., Dadgar, J., Fischbach-Weiss, S. C. and Messing, R. O. (2013). Prkcz null mice show normal learning and memory. Nature 493(7432): 416-419.
  7. Lee, I., Hunsaker, M. R. and Kesner, R. P. (2005). The role of hippocampal subregions in detecting spatial novelty. Behav Neurosci 119:145-153.
  8. Lesburguères, E., Sparks, F. T., O’Reilly, K. C. and Fenton, A. A. (2016). Active place avoidance is no more stressful than unreinforced exploration of a familiar environment. Hippocampus 26(12): 1481-1485.
  9. Mumby, D. G., Gaskin, S., Glenn, M. J., Schramek, T. E. and Lehmann, H. (2002). Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts. Learn Mem 9(2): 49-57.
  10. Norman, G. and Eacott, M. J. (2005). Dissociable effects of lesions to the perirhinal cortex and the postrhinal cortex on memory for context and objects in rats. Behav Neurosci 119:557-566.
  11. Steckler, T., Drinkenburg, W. H., Sahgal, A. and Aggleton, J. P. (1998a). Recognition memory in rats--I. Concepts and classification. Prog Neurobiol 54(3): 289-311.
  12. Steckler T, Drinkenburg, W. H., Sahgal, A. and Aggleton, J. P. (1998b). Recognition memory in rats--II. Neuroanatomical substrates. Prog Neurobiol 54:313-332.
  13. Tsokas, P., Hsieh, C., Yao, Y., Lesburgueres, E., Wallace, E. J., Tcherepanov, A., Jothianandan, D., Hartley, B. R., Pan, L., Rivard, B., Farese, R. V., Sajan, M. P., Bergold, P. J., Hernandez, A. I., Cottrell, J. E., Shouval, H. Z., Fenton, A. A. and Sacktor, T. C. (2016). Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice. Elife 5: e14846.


该方案最初设计用于检查PKMζ敲除(即PKMζ-无效)小鼠的长期空间记忆(Tsokas等,2016)。我们的主要目标是测试这些动物是否维持先前获得的空间信息的能力对于需要记住的空间信息的类型和复杂性是敏感的。因此,我们修改并组合成一个单一协议,三个新颖性偏好测试,特别是上下文中的对象,现场对象和位置对象测试,改编自以前的啮齿动物研究(Mumby等, 2002; Langston and Wood,2010; Barker and Warburton,2011)。在该过程的训练(学习)阶段期间,小鼠被重复地暴露于三个不同的环境中,在这三个不同的环境中,它们学习环境特定的一组不相同对象的空间排列。在这个学习阶段完成后,每个鼠标接收三个不同的内存测试,配置为环境不匹配,其中以前学习的空间内对象配置已经从原始的训练情况进行了修改。由于被操纵的空间关联类型,专门评估对象上下文和对象位置关联的记忆,错配测试在认知需求上有所不同。在每次记忆测试期间,用于探索小说(错放的)和熟悉的对象的时间差被计算为新奇性歧视的指标。该索引是先前获取的空间关联的记忆回忆的行为测量。
新颖性偏好任务易于使用,为调查认知功能(如啮齿动物空间记忆)(Kinnavane等,2015)提供了极大的灵活性。以前的研究启发了以前的研究,这些研究已经使用了新颖性偏好范例的不同空间版本,特别是就地对象,对象上下文和对象位置任务变体(Mumby等人,2002; Langston和Wood,2010; Barker and Warburton,2011)。通常,这些任务协议包括短暂的学习阶段,随后是存储器保留测试,在此期间,以特定方式修改环境的原始空间配置。然而,在以前的研究中,不同的任务变体已经单独呈现,需要对独立的动物组进行测试。此外,测试通常是短暂的延迟(几分钟到几个小时),而不是几天,这是专门评估长期记忆持久性机制所要求的。在本实验设计中,执行每个记忆保留测试以评估在相同动物中持续至少24小时的记忆,并且对应于原始学习体验的特定空间操纵。另外,通过改变在学习阶段(即4个对象与2个对象)中呈现的对象的数量,以下协议允许直接操纵要被记住的信息量,而不仅仅是信息的类型。
在这里,我们提出最近用于评估使用野生型和PKMζ敲除小鼠24小时长期记忆持久性的分子机制的行为方案(Tsokas等,2016)。该协议是足够敏感的,以揭示对四个对象之间的对象间关联至关重要的分子机制需要持续激酶PKMζ,而非PKMζ依赖机制足以维护上下文中对象关联和对象涉及两个对象的位置关联。在背部海马水平也观察到支持对象现场和对象上下文关联的神经机制之间的类似解离。特别是,背部海马的永久性或临时性损伤足以损害获得现场对象关系而不是上下文关联关系(Langston和Wood,2010; Langston等人,2010年综述),具有潜在的齿状回CA3子电路的更大作用(Lee等,2005)。对象上下文关联似乎在很大程度上取决于大脑皮层功能(Norman和Eacott,2005),而对象位置关联往往对海马操作敏感(Hardt等人,2010; Barker和Warburton,2011)。虽然这些分离表明认知功能的敏锐依赖性,每个测试评估不同的大脑区域,但这些结构 - 功能关联的边界可能不是确定的,因为病变的后果取决于任务参数,包括语境线索是局部的还是远端的以及是否通过记忆采集和整合测试或记忆保持测试来评估结构的作用(由Langston等,2010审查)。实际上,识别记忆,其中一个子集由我们在这里描述的对象上下文位置范例评估

关键字:空间, 新奇偏好, 歧视记忆, 识别记忆, 环境


  1. 3个打开的盒子(31 x 31 x 19 cm,长x宽x高)。它们是从Thoren Caging Systems(Hazelton,PA)
  2. 3套不同的层压板,在白色背景上显示黑色图案(见图1)
  3. 10个不一致的对象
    注意:我们使用了塑料玩具(美国PetCo ®)的组合,不同咀嚼形状和/或材料不同的烧瓶和罐子,如强天然橡胶,派雷克斯聚丙烯和铝。这些是小鼠探索的对象。每个物体都是独一无二的,但大小相等,它们足够高,可以防止老鼠爬上物体。足迹尺寸约6厘米,高度为17厘米。物体必须是可拆卸的,应该很容易清洗(参见图1)
  4. 野生型雄性成年(4月龄)小鼠(C57BL6/J,THE JACKSON LABORATORY)
    1. 在本方案的原始出版物中,我们还使用来自PKMζ敲除小鼠系的成年突变体雄性小鼠(PKMζ-null,最初描述于Lee等人,2013),其由罗伯特O提供的育种者繁殖Messing(德克萨斯大学,德克萨斯州奥斯汀分校)。
    2. 在12-12小时的明暗循环下,将小鼠单独饲养在具有可控温度(23℃)和湿度的环境中,随意获得食物和水。所有行为程序均在周期的7 AM-7PM光照阶段进行。虽然没有进行测试,但只要住房和培训条件一致,反向光周期的培训和测试对行为结果不应有影响。将小鼠单独饲养在双笼中(每笼2个单独的隔间;每笼1个野生型和1个敲除)。突变动物在我们的设施繁殖,当动物断奶时,基因型是已知的。研究中的其他小鼠单独饲养,因为它们植入可被另一只小鼠损伤的脑内注射套管或微电极阵列。因此,我们选择个人住房来确保所有动物之间类似的住房条件。替代住房,例如成对的,可能是实际的。
  5. 水和70%EtOH


  1. 仪器


  2. 对象
  3. 配备½"4-12 mm CCTV镜头(TAMRON,目录号:12VM412ASIR)和软件(Tracker,Bio-Signal Group)的高架摄像机(Firefly USB 2.0摄像头,FLIR Integrated Imaging Solutions,目录号:FMVU-03MTM-CS) ,Acton,MA),用于数字视频跟踪鼠标的位置并记录视频的整体行为
  4. 跟踪系统
    我们使用PC控制的视频跟踪系统(Tracker,Bio-Signal Group,Acton,MA)在行为过程中准确检测动物在每个环境中的位置,并记录其离线分析的探索性行为。在每1/30秒的视频帧中,鼠标的位置被视为其图像的质心。


  1. 在行为实验前一周,每天,所有动物都熟悉从住房位置到实验室的运输程序。动物也由实验室在实验室处理,以便他们习惯于该程序。
  2. 每天,在适应行为实验开始前30分钟,将小鼠置于实验室中。
  3. 整个行为过程的持续时间约为9天。如图2所示,每个鼠标首先在对象上下文位置任务中进行训练和测试,然后是对象位置任务。每个任务过程包括三个阶段:预培训,培训和保留测试。


    1. 第1天:前后训练A和B
      1. 动物习惯于背景A和B.他们探索每个盒子10分钟,没有物体存在。每个预训练会话间隔1小时。在动物被放置在盒子的中心,其鼻子面向南墙之后,预训练会立即开始。
      2. 在每个预培训课程之间,盒子和物体用水洗涤,然后用70%EtOH清洗,这样可以使其干燥。这样做是为了防止嗅觉线索的建立。
      3. 在10分钟的探索结束时,将动物从上下文中移除并返回其家笼。
      注意:在Tsokas等人的情况下,每个实验组中的动物之间的背景曝光顺序是平衡的,这是基因型。 (2016)。 
    2. 从第2天到第4天:在A和B的情况下训练

      表1.平衡上下文探索的顺序示例( ie 。,上下文A与B)培训日与在原始研究中,每个实验组( ie ,Wild Type vs.PKMζ-null)中的顺序

    3. 第5天:保留测试#1
      给予小鼠第一个内存保留测试,它是对象/上下文或对象/位置不匹配测试。在对象/上下文不匹配测试中,仅在一个上下文中遇到的四个对象中的两个被放置在第二个上下文中,而在对象/位置不匹配测试中,来自一个四对象配置的两个对象被放置在他们以前遇到的相同的上下文。允许动物探索3分钟。保留测试会话的3分钟持续时间已被仔细验证。 3分钟后,小鼠变得熟悉新颖的对象配置,因此动物不再检测到由物体排列触发的空间新颖性。随后,小鼠将会平等地探索所有的对象,无论是否存在不匹配
    4. 第5天:再曝光
      同一天,在记忆保留测试后的一个小时,小鼠接受额外的训练(在每个上下文A和B中进行5分钟的探查两次),以便最小化学习可能具有的失配对象 - 空间关联的潜在影响被保留试验诱导。
    5. 第6天:保留测试#2

      图3.平衡对象和/或放置排列和保留测试顺序。 A.通过在对象/位置和对象/上下文不匹配测试中进行对象对排列来实现空间对象平衡。因为每个主题可能对特定对象和/或上下文具有特殊的偏好或不喜欢,所以在记忆测试期间平衡每个组内的对象之间的对象上下文呈现是重要的。有很多的排列可能。考虑通过沿水平,垂直和两个对角线交换物体对来随机化上下文中的对象,以生成六个独特的排列模式,并从每个上下文交换一半对象,从而生成12个对象上下文布置。上下文A中从初始训练配置(训练配置显示为a-b-c-d)表示的对象排列的六个示例是b-a-c-d(水平),a-d-c-b(垂直),a-c-b-d(对角线)它们说明了对象/位置不匹配测试的空间操纵的平衡。配置e-f-c-d(水平),a-f-c-h(垂直),a-f-g-d(对角线)示出了对象/上下文不匹配测试的平衡空间操纵的示例。 B.在第5天和第6天(,对象/位置与对象/上下文不匹配测试)之间平衡保留测试顺序的示例以及每个实验组内动物之间的对象排列类型(例如,,Wild Type vs.PKMζ-null)。

    6. 第7天:上下文中的预培训C
    7. 第八天:上下文中的培训C
    8. 第9天:保留测试#3
    9. 记忆性能的行为测量
      每个视频都被离线分析,以手动评估鼠标对每个保留测试的对象的探索时间。对象探索被定义为动物朝向物体的鼻子,距离< 2厘米。因此,每个视频都会在跟踪器软件中使用围绕每个对象的2厘米宽的环形掩模进行重放,以定义对象探索区域。在这个区域内,只有当动物的鼻子朝向物体时,动物的活动如用爪子嗅探或触摸物体才算作物体探索活动(见注释)。测量对象探索由对动物实验组无视的实验者进行,物体是否被改变。在三个不同的记忆测试中的记忆性能被量化和分析,使用的差异指数被计算为探索改变的(,不正确,错放或重新定位)对象所花费的时间的绝对差异,在花费时间的时间探索所有的对象。因此,该指数考虑到勘探总量中的个体差异。良好的记忆保留率对应于正面的辨别指标,反映出动物花费更多的时间来探索不正确的(物体/上下文不匹配),位移(物体/位置不匹配)或重新定位(物体/位置不匹配)保持不变(图4)。





  1. 为了使每个要被探索的对象都是唯一和可识别的,我们建议使用各种形状和类型的材料。在实验之前,应该对实验对象的选择进行验证,在试点研究中用鼠标测试每个配置集,以避免明显偏向于调查一个对象。
  2. 动物不应该爬上物体,因为它会影响探测活动的测量精度。
  3. 物体的照明应该是同质的,以避免在实验箱的角落处产生阴影。暗角通常是老鼠优选的,因此优选场所的物体偏好的存在可能引起可能干扰动物整体探索活动的地方偏好。
  4. 一个安静,昏暗(10-15勒克斯)实验室是小鼠自发探索行为的首选环境。
  5. 在行为实验的每一天结束时,动物都被不受干扰地保持一小时,然后被运回到动物住所。
  6. 使用视频跟踪系统记录所有行为会话。虽然对象探索活动是一种行为测量,只能通过给定环境中存在的对象进行评估(在训练和记忆保留测试期间),在第1天的预训练期间执行一般运动活动测量和动物跟踪以评估组移动和一般探索活动的差异,其差异可以将新颖性偏好的评估偏向于保留测试。


该行为协议最初用于Tsokas等人,2016年。这项工作得到NIH授权R21NS091830和R01MH099128(AAF)和R37MH057068,R01MH53576,R01DA034970和Lightfighter Trust(TCS)的支持。


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Copyright Lesburguères et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lesburguères, E., Tsokas, P., Sacktor, T. C. and Fenton, A. A. (2017). The Object Context-place-location Paradigm for Testing Spatial Memory in Mice. Bio-protocol 7(8): e2231. DOI: 10.21769/BioProtoc.2231.
  2. Tsokas, P., Hsieh, C., Yao, Y., Lesburgueres, E., Wallace, E. J., Tcherepanov, A., Jothianandan, D., Hartley, B. R., Pan, L., Rivard, B., Farese, R. V., Sajan, M. P., Bergold, P. J., Hernandez, A. I., Cottrell, J. E., Shouval, H. Z., Fenton, A. A. and Sacktor, T. C. (2016). Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice. Elife 5: e14846.