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Jun 2018

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An Alternative Maze to Assess Novel Object Recognition in Mice
一种评价小鼠新目标识别的替代迷宫   

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Abstract

The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior. This memory test, being performed in a two-arms maze, is suitable for several strains of mice (including inbreed and outbreed) and does not require extended training sessions allowing an accurate temporal assessment of memory formation. This particular maze increases the mouse exploration time and reduces variability compared to other arenas used before to assess NOR. As both long- and short-term NOR memory can be easily and accurately quantified using this paradigm, this improved methodology can be easily applied to study pharmacological, genetic or age-related modulation of cognitive function.

Keywords: Novel Object recognition memory (新目标识别存储记忆), Cognition (认知), Mouse behavior (小鼠行为), Memory task (记忆任务), Learning (学习行为), Animal behavior (动物行为)

Background

Novel object recognition (NOR) memory task is a commonly used experimental behavioral task aimed at studying learning and memory (Ennaceur, 2010). The main postulate behind this behavioral task is that, in the presence of a novel and a previously presented (familiar) object, rodents increase their exploration towards the novel object (Ennaceur and Delacour, 1988). The increased exploration of the novel object is interpreted as indirect evidence that animals acquired a memory of the familiar object, and thus increase their exploration to the novel one. As a consequence, NOR paradigm is considered a reliable model to test hippocampal and temporal lobe function, as lesions within these brain regions abolish recognition memory (Winters et al., 2008; Broadbent et al., 2010).

There are several advantages of using the NOR task. First, NOR task takes advantage of the animal’s tendency to approach and explore novelty. Therefore, this task does not require preliminary extended training and training can happen in a single trial session, allowing a robust temporal definition of the different stages of the memory formation (i.e., acquisition, consolidation, re-consolidation and retrieval). Also, it does not require exposure to aversive or stressful stimuli stronger than novelty itself, nor it requires food or water restriction. Altogether, these factors have contributed to the growing popularity of this behavioral paradigm (Dere et al., 2007).

The NOR task has been replicated using a variety of environmental designs and objects in rodents. This task has been classically performed in a large open field where rodents navigate to explore the objects (Bevins and Besheer, 2006) or in a Y-shaped maze with very short arms were rats are exposed to two objects that they explore without the need of extensive exploration (Winters et al., 2004). NOR tasks that involve an open field environment require longer habituation periods (several days) that must be performed during several consecutive training sessions (Dere et al., 2007). As a consequence, the evaluation of long-term NOR memory seems less reliable (Sik et al., 2003). One important limitation of the open field setting is increased variability within similar experimental groups observed in terms of the interaction/exploratory time of the animal with the object. The high variability can be caused by spatial and contextual confounds together with the pro-anxiogenic effects of the environment (open field) (Hale et al., 2008). Overall, this lengthens the overall assay, reduces the accuracy of the test and entails the use of a higher number of animal in each experimental group. On the other hand, the Y-shaped maze used with rats, hinders the assessment of the locomotor activity of the animals, which in turn could bias the outcome of the test due to the short length of the corridors where the object exploration takes place.

Here, we propose a maze consisting in two arms separated by 90 degrees that has been equally called in previous publications as L- or V-Maze (Puighermanal et al., 2009 and 2013; Busquets-Garcia, et al., 2011, 2013, 2016 and 2018; Aso et al., 2012; Hebert-Chatelain et al., 2016; Aloisi et al., 2017). This maze has long corridors to minimize the context surrounding the objects and to reduce the weight of other possible cues rather than the objects themselves. These features direct the animal’s exploratory behavior towards the objects and facilitates the interaction of the animal with the object. Moreover, the arms in this maze are relatively narrow, thus reducing the possible anxiety-related bias of the open field environment. Altogether, this version of the maze maximizes the exploration time of the objects relative to the surrounding context and increases the accuracy of the test. This improved setting has been now successfully used to evaluate short- and long-term memory depending on the retention time defined between the training session and the test session (3 h and 24 h, respectively) in both inbreed (C57BL/6) and outbreed (CD-1) mice (Puighermanal et al., 2009 and 2013; Aso et al., 2012; Busquets-Garcia et al., 2011, 2013, 2016 and 2018; Hebert-Chatelain et al., 2016; Aloisi et al., 2017). Also, it improves both the variability of the assay and the reproducibility, which in turn reduces the number of mice required, the length of the sessions and the overall complexity of the protocol. However, this new NOR protocol have the limitation that it has to be adapted (e.g., light intensity, room noise, room space or objects used) every time that it is used in a novel mouse strain or when it is set up in a new animal space. Thus, it needs a pilot experiment to verify all these parameters and it might not be as flexible as an open field arena. Sor far, this specific NOR task has been used in access memory performance in a mouse model of Alzheimer (8 months or 18 months) (Aso et al., 2012), to study the acute effects of stress on recognition memory (Busquets-Garcia et al., 2016), the memory deficits in a mouse model of Fragile X syndrome (Busquets-Garcia et al., 2013; Aloisi et al., 2017) and the modulation of memory by the endocannabinoid system (Puighermanal et al., 2009 and 2013; Hebert-Chatelain et al., 2016; Busquets-Garcia et al., 2018; Robin et al., 2018; Oliveira da Cruz et al., 2019).

Materials and Reagents

  1. Disposable paper towels
  2. Mice: 8-12 weeks old C57BL/6J and/or CD1 mice (Charles River, France or any company that provide mice for laboratories)
  3. Water
  4. 70% ethanol

Equipment

  1. The NOR maze (see Figure 1)
    We have designed and built this maze in the lab. The material used to build the maze was black or dark grey matt plexiglas.


    Figure 1. Maze to assess NOR memory. Dimensions are in cm.

  2. Two pairs of objects (see below for information regarding the objects)
    These pairs of objects must be first validated in mice (see Procedure) to guarantee no intrinsic preference or aversion.
  3. Experimental room with adjustable light (30-50 lux)
  4. Real-time video acquisition system
  5. Casual Stopwatches or any computer application to count the exploration time

Software

  1. Any video Acquisition Software
  2. Behavior Scoring Panel-(c) 2008 by A. DUBREUCQ Version 3.0 beta. A home-made software that allow us counting the exploration time.
    Note: If possible and to avoid any confounding factor, it is recommended that the experimenter is not present in the same room where the mouse is performing the task.

Procedure

  1. Habituation session
    This step is performed to familiarize mice with the maze. The habituation session lasts for 9 min.
    1. Turn on the video recording system and set the parameters for data acquisition: Make sure that there is constant uniform lightning conditions in the maze (areas of interest, contrast for mouse detection, etc.) and in the room throughout the session (habituation, training and test sessions).
    2. Place the animal in the maze without objects at the starting point (crossing-point between both arms, Figure 1).
    3. At the end of the session, remove the animal from the maze and return it to its home-cage.
    4. Then, clean maze and the objects with 70% ethanol and move to the next animal (back to Step A2).
      Note: In order to avoid that odor cues from previous mice alter mouse exploration, it is crucial to carefully clean the surface of the maze and objects between each session with a 70% ethanol solution.

  2. Training session
    The training session is performed 24 h after the habituation session. The training session lasts for 9 min.
    1. Turn on the video recording and data acquisition system.
    2. Place two identical objects in the extremities of the maze in direct contact with the wall (see Figures 1 and 2).


      Figure 2. Scheme of the object recognition protocol. There are three different sessions (habituation, training and test) and each session lasts 9 min. Short- or long-term memory can be assessed depending on the inter-session interval between the training and the test session.

    3. Place the animal at the starting point facing towards the edge of the two arms (crossing-point between the two arms).
    4. Use the stopwatches, to record the time that the mouse spends exploring each object (see below). During this session, rodents usually spend around 50% of the total exploratory time for each one of the objects.
    5. At the end of the training session, remove the mouse from the maze and leave it undisturbed in its home-cage.
    6. After cleaning the maze as explained in the “habituation session” of the maze, start the training session for the following animal.

    Pharmacological treatments
    Pharmacological treatments can be used to study cellular and molecular mechanisms involved in different memory phases of NOR. For instance, in order to test the effect of a particular pharmacological treatment on memory acquisition (Figure 2), the drug must be administered before the training session. The administration time must be adjusted by taking in consideration the pharmacokinetics of the substance. Moreover, it is important to verify that the drug has no locomotor effects as it may influence the task outcome independently of the memory performance (e.g., impaired locomotor activity may affect exploration and consequently the capacity of the animal to move towards the objects to explore them). To study the effect of a pharmacological treatment on memory consolidation, the drug should be administered after the training session. Finally, to assess memory re-consolidation, mice are re-exposed to the same training conditions (same combination of objects during a 9 min session) 24 h after the first training session and the drug can be administered before or after the second training session.

  3. Testing phase
    The test session could be performed 3 h after training to evaluate short-term memory, or 24 h after training to evaluate long-term memory. The training session lasts for 9 min.
    1. Turn on the video recording and data acquisition system.
    2. Clean the maze.
    3. Place one of the familiar objects at the end of one arm and the novel object at the end of the other arm. The position of the novel object (i.e., right vs. left arm) must be counterbalanced in between mice to avoid any possible confounding cue in the room that results in a preference for one of the arms of the maze.
    4. Place the animal at the starting point, as previously mentioned above.
    5. As in the previous sessions (habituation and training sessions), the observation is accomplished by a closed-circuit camera attached to a recording system to avoid unnecessary stress or uncontrolled cues to the animal (see Video 1).

      Video 1. Representative video showing object exploration in both arms of the maze

    6. Use two stopwatches and assign each stopwatch to record the exploration time for each object. The experimenter, blind to the experimental conditions, measures the exploration time for each object analyzing the image obtained by the closed-circuit camera. Simultaneously, the video is recorded for documentation purposes together with video-tracking of general activity (time spent in each arm, number of entries in each arm and distance travelled).
    7. At the end of the test session, remove the animal from the maze and return it to the home-cage. After cleaning the maze with water, start with another animal (back to Step C3).
    8. After the session, export the experimental data and analyze the results (see Data analysis).

    Note: If the total exploration time is very low (< 5-10 s) in the training and/or test sessions the results of these mice are not considered for the experiment. This may happen due to:
    1. The object is not adequate for the task (i.e., it may induce stress or anxiety to the animals). Verify that the object was properly cleaned as it could contain the odors of a previous animal and re-evaluate the objects to understand if there is intrinsic preference or aversion.
    2. The animal does not explore the maze because of a locomotor deficit or it does not explore the maze due to a pharmacological treatment and/or a genetic manipulation. If there is a clear locomotor alteration during the performance of the task, exclude the animal from the analysis.

Data analysis

  1. Quantification of object exploration
    Exploratory time is scored manually by an experimenter blind to the experimental design during the training and test sessions. The manual scoring is preferred due to the current difficulty for automated systems to accurately discriminate between tail and head and to correctly count exploration time. Nevertheless, we acknowledge that deep-learning based approaches for pose estimation (Mathis et al., 2018; Nath et al., 2019) may provide a reliable way to automatize exploration quantification in the near future.
    Exploration of an object is scored when: the animal nose is facing directly the object (< 2 cm) as described before (Robin et al., 2018; Oliveira da Cruz et al., 2019). Exploration is not scored when: the animal does not interact with the object or it stands on the top of the object without facing the object.
    Note: If the animal does not interact with both objects at least once during the complete duration of the session, the session is not valid.

  2. Analysis of Object Exploration
    In order to evaluate differences in the exploration during this task when comparing two or more experimental groups, 2 types general analysis can be performed: 1) Discrimination Index (DI); 2) Familiar vs. Novel analysis. In addition, the total exploration time is also expressed to control variability in overall exploration between experimental groups. The DI is calculated with the following formula:
    DI = [Timenovel – Timefamiliar)]/[(Timefamiliar) + (Timenovel)]

    Additional variables can be measured: time spent in each arm, number of entries in each arm and distance travelled. Exploration data can be represented in mean ± SEM. Two-way Student’s t-test and one-way ANOVA statistical analysis are the statistical tests used in these experiments. Comparisons are considered statistically significant when the level of significance is lower than 0.05.

  3. Expected Results
    In the experimental conditions reported in this protocol, the general exploration time observed is around 15-40 s for each object during a typical training session. If the mice discriminate between the familiar and the novel object, the exploration time in the test session will be around 10-35 s for the familiar object and around 30-60 s for the novel object (Figure 3 and Table 1). If the recognition memory is impaired, as we described in Tetrahydrocannabinol (THC)-injected mice (Puighermanal et al., 2009), mice will explore the same amount of time both novel and familiar objects during the test session (20-40 s), and so they will not discriminate between them (Figure 3 and Table 1).


    Figure 3. Representative graph of an example of the NOR task. Further details in Table 1.

    Table 1. An example of the expected data and analysis. Representative example of an experiment using a pro-amnesic dose of delta9-tetrahydrocannabinol (THC, 10 mg/kg, i.p., light colour) in CD1 and C57BL/6J mice, or its vehicle (VEH, dark colour) as control, administered after the training session. The exploration time (seconds) in the familiar object (F); the exploration time on the second object in the training session (F’); the exploration time in the novel object (N); the total exploration time (ET = F + F’ or ET = F + N); the percentage of exploration time devoted to each object during the training and the test sessions (% ET = [ F/ET ] x 100; % ET = [F’/ET] x 100; %ET = [N/ET] x 100); the discrimination index is calculated for the test session (DI = [N – F]/[N + F]).

Notes

  1. Mice are housed for a minimum of 5 days in the experimental room where the behavioral task is set to take place. Animals are commonly housed collectively (3-6 animals per cage) in a saw dust environment with nesting material (cotton cube), water/food ad libitum and controlled temperature (21 ± 1 °C) and humidity (55 ± 10%). Lighting is maintained at 12-h dark/light cycles (on at 7:00 a.m. and off at 7:00 p.m.) and experiments take place in the beginning of the light phase (typically 9:00 a.m.-2:00 p.m.). Home-cages should not be changed, and animals should not be disturbed during the previous days preceding the task as it may affect their performance. Housing facility should be located in the vicinity of the testing rooms as the stress and novelty of transport may alter exploration.
  2. Choosing suitable objects is an essential step in this task. At least two distinct pairs of objects are needed. Features such as texture (stone, wood, plastic, metal) (Figure 4), color tonality and shape should be carefully considered when selecting objects to be used in this task. It is important to verify that animals spend a similar amount of time interacting with each object in the training session. Any preference or aversion for the objects should be avoided since it could represent an important bias for the interpretation of the results. In order to avoid this possible bias, preliminary experiments must be performed presenting to the animals both objects simultaneously and analyzing the exploration time for each one. The time exploring each object should be similar (around 50% of the total exploration time devoted to each object) when both objects are presented for the first time to the mice. The exploration time for each object can be influenced by the shape and physical characteristics of the object. However, objects must be different enough from each other (color tonality, texture, shape) to facilitate the discrimination between both objects in the test session.


    Figure 4. Objects used in our NOR paradigm. In this image you can find different examples of the objects used in our NOR paradigm. As you can see, different colors, shapes and materials can be used.

  3. It is important to avoid odor cues and it is therefore mandatory to clean the maze and objects between animals and especially between experiments using different experimental conditions.
  4. It can happen that control animals do not show appropriate discrimination of the novel object. This can be due to a specific preference for one object or it could also be caused by the different, uncontrolled, conditions between the training and test sessions (e.g., animals were disturbed by animal caretaker during the period between the training and test sessions) or by olfactory cues left by other mice and not well cleaned in between animals and/or sessions.
  5. Very rarely, the mouse can jump out of the maze. To prevent these events, a transparent plastic cover can be placed above the maze.

Acknowledgments

We thank Delphine Gonzales, Nathalie Aubailly, and all the personnel of the Animal Facility of the NeuroCentre Magendie for mouse care as well as the personnel from the Parc de Recerca Biomèdica de Barcelona (PRBB). We would also like to thank the people who have performed experimental work using this task. This work was supported by INSERM (to G.M.), EU–FP7 (FP7-PEOPLE-2013-IEF-623638 to A.B.-G.), French State/Agence Nationale de la Recherche (ANR-10-IDEX-03-02 to A.B-G), and MINECO from AEI (RYC-2017-21776) to A.B-G.

Competing interests

The authors declare no competing interests.

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简介

[摘要] 新颖对象识别(NOR)任务是一种行为测试,通常用于评估情节式声明性记忆,它依赖于啮齿动物探究新颖性的先天趋势,在这里,我们介绍一种用于评估小鼠NOR记忆的迷宫,通过增加探索性行为,减少了测试时间,同时提高了测量的可靠性。此记忆测试是在两臂迷宫中进行的,适用于多种小鼠品系(包括近交和近交),不需要进行长期培训。允许的记忆形成一个准确的时间评估会话。这种特定迷宫增加鼠标勘探时间,降低变性相比之前用于评估NOR其他领域。由于长期和短期的NOR存储器可以容易地和准确地屈antified使用此范式上,这种改进的方法可以轻松地应用于研究认知功能的药理,遗传或与年龄相关的调节。

[背景] 新型对象识别(NOR)记忆任务是一种旨在研究学习和记忆的常用实验行为任务(Ennaceur,2010),该行为任务背后的主要假设是,在存在小说和先前提出的情况下(熟悉的)物体,啮齿动物增加了对新物体的探索(Ennaceur and Delacour,1988)。对新物体的增加探索被解释为动物获得了对熟悉物体的记忆的间接证据,从而增加了对新物体的探索。因此,NOR范式被认为是测试海马和颞叶功能的可靠模型,因为这些大脑区域内的病变消除了识别记忆(Winters 等,2008;Broadbent 等,2010)。

使用NOR任务有几个优点:首先,NOR任务利用了动物接近和探索新奇的倾向,因此,此任务不需要进行初步的扩展训练,并且可以在单个试验阶段进行训练,从而使健壮的时间定义记忆形成的不同阶段(即获取,巩固,重新巩固和恢复)。此外,它不需要接触比新颖性更强的厌恶或压力刺激,也不需要食物或水的限制。这些因素促成了这种行为范式的日益普及(Dere 等,2007)。

NOR任务已在啮齿动物中使用了多种环境设计和物体进行了复制,该任务通常在大型空旷地中进行,啮齿动物会导航以探索物体(Bevins和Besheer,2006),或者在Y型迷宫中老鼠的手臂非常短,它们无需进行大量探索就可以暴露于他们探索的两个物体中(Winters 等人,2004年),而涉及野外环境的NOR任务则需要更长的适应时间(数天),必须在数个时间内执行连续训练(Dere et al。,2007 )。因此,长期NO R记忆的评估似乎不那么可靠(Sik et al。,2003 )。开阔地域设置的一个重要限制是在相似条件下的可变性增加。根据动物与物体的相互作用/探索时间观察实验组。高变异性可能是由空间和上下文混淆以及促焦虑作用引起的 (Hale et al。,2008)。总体而言,这延长了整个检测的时间,降低了测试的准确性,并在每个实验组中使用了更多的动物。老鼠使用的Y形迷宫阻碍了对动物运动能力的评估,由于进行物体探索的走廊较短,反过来可能会使测试的结果产生偏差。

在这里,我们建议由在相隔90度的两个臂迷宫已经被同样称为以前的出版物如L-或V-迷宫(Puighermanal 等人,2009年和2013年; 布斯克茨-加西亚等人,2011,2013,2016和2018; Aso 等人,2012 ; Hebert-Chatelain 等人,2016 ; Aloisi 等人,2017 )。这种迷宫有很长的走廊,以最大程度地减少物体周围的环境并减轻其他可能线索的重量这些特征而不是物体本身,这些特征将动物的探索行为引向物体,并促进了动物与物体的相互作用。此外,该迷宫中的手臂相对较窄,从而减少了空旷地区可能与焦虑相关的偏见。总而言之,此版本的迷宫可以最大程度地延长物体相对于周围环境的探索时间,并提高测试的准确性。此改进的设置现已成功用于评估短期和长期记忆 取决于近交(C57BL / 6)和近交(CD-1)小鼠在训练和测试之间定义的保留时间(分别为3小时和24小时)(Puighermanal et al。,2009和2013; Aso 等人,2012年 ;Busquets-Garcia 等人,2011年,2013年 ,2016年和2018年;Hebert-Chatelain 等人,2016年;Aloisi 等人,2017年)。此外,它还改善了测定的可变性和可重复性,从而减少了所需的小鼠数量,会话时间和协议的整体复杂性。但是,此新的NOR协议具有必须适应的限制(例如,光强度,房间噪声,房间空间或使用的物体)每次在新颖的小鼠品系中使用时或在新的动物空间中使用时,都需要进行先导实验以验证所有这些参数,并且可能不如开放式系统灵活现场arena.Sor 远,这个特定的NOR任务已在存取存储器的性能适用于老年痴呆症的小鼠模型 (8个月或18个月)(Aso 等,2012),研究应激对识别记忆的急性影响(Busquets-Garcia 等,2016),脆性X综合征小鼠模型(Busquets)的记忆缺陷-Garcia 等人,2013;Aloisi 等人,2017)以及内源性大麻素系统对记忆的调节作用(Puighermanal 等人,2009和2013;Hebert-Chatelain 等人,2016;Busquets-Garcia 等人, 201 8 ; Robin 等人,2018 ; Oliveira da Cruz 等人,2019 )..

关键字:新目标识别存储记忆, 认知, 小鼠行为, 记忆任务, 学习行为, 动物行为

材料和试剂


 


一次性纸巾
小鼠:8-12周大的C57BL / 6J和/或CD1小鼠(法国查尔斯河或提供实验室小鼠的任何公司)

70%乙醇
 


设备


 


NOR迷宫(请参见图1)
我们在实验室中设计并建造了这种迷宫,用来建造迷宫的材料是黑色或深灰色的亚光有机玻璃。


 


D:\ Reformatting \ 2020-4-7 \ 1902471--1424 Giovanni Marsicano 652762 \ Figs jpg \ Fig1.jpg


图1.评估NOR记忆的迷宫尺寸以厘米为单位。


 


两对对象(有关对象的信息,请参见下文)
Ť HESE对对象都必须在小鼠中(见步骤)首先被验证,以保证没有内在的偏好或厌恶。


可调光实验室(30-50 lux)
实时视频采集系统
休闲秒表或任何计算机应用程序来计算探索时间
 


软件


 


任何视频采集Softwar Ë
行为评分面板- (c)2008年,A。DUBREUCQ版本3.0 beta。这是一种家用软件,可以让我们计算探索时间。
注意:如果可能并为了避免造成任何混淆因素,建议不要将实验人员放在鼠标正在执行任务的同一个房间中。


 


程序


 


习性会议
执行此步骤以使小鼠熟悉迷宫。习惯化过程持续9分钟。


打开视频记录系统并设置用于数据采集的参数:确保在整个会话过程中(迷宫,训练等)在迷宫(感兴趣的区域,用于鼠标检测的对比度等)和房间中始终存在恒定的均匀闪电条件。和测试会话)。
在起点处(两臂之间的交叉点,图1)将动物放在没有任何物体的迷宫中。
在会议结束时,将动物从迷宫中移出并放回笼中。
然后,用70%的乙醇清洁迷宫和物体,然后移至下一只动物(返回步骤A2)。
注意:为了避免以前的老鼠发出的气味提示改变老鼠的探索,每次使用70%乙醇溶液仔细清洁迷宫和物体的表面至关重要。


 


培训课程
习惯性训练后24小时进行训练,持续9分钟。


打开视频记录和数据采集系统。
将两个相同的物体放置在迷宫的末端,与墙壁直接接触(见图1 和2 )。
 


D:\ Reformatting \ 2020-4-7 \ 1902471--1424 Giovanni Marsicano 652762 \ Figs jpg \ Fig 2-word.jpg


2.图计划目标识别协议。有三种不同的会话(习惯化,训练和测试),并且每个会话持续9分钟短期或长期记忆可以评估取决于会话间间隔培训和之间测试会话。


将动物放在面向两只手臂边缘的起点(两只手臂之间的交叉点)上。
使用此秒表记录鼠标花费在探索每个物体上的时间(见下文)。在此期间,啮齿动物通常花费每个物体探索总时间的50%左右。
在训练结束时,将鼠标从迷宫中移开,并将其放在笼子中不受干扰。
按照迷宫的“习惯性练习”中的说明清洁迷宫后,开始对以下动物进行训练。
 


药理治疗


药理学治疗可用于研究NOR的不同记忆阶段所涉及的细胞和分子机制,例如,为了测试特定药理学治疗对记忆获得的影响(图2 ),必须在训练之前使用该药物。必须考虑到该物质的药代动力学来调整给药时间。此外,重要的是要验证该药物是否具有运动作用,因为它可能独立于记忆力而影响工作结果(例如运动能力受损)为了研究药物治疗对记忆巩固的影响,应在训练后服用该药物,最后评估记忆的巩固,将老鼠重新置于相同的训练条件下(9分钟内对象的相同组合)24 在第一个训练课后h,可以在第二个训练课之前或之后给药。


 


测试阶段
该测试课程可以在训练后3小时进行以评估短期记忆,或在训练后24小时进行以评估长期记忆。该训练持续9分钟。


打开视频记录和数据采集系统。
清洁迷宫。
放置一个熟悉的物体在最后的一个臂和新物体在最后的另一个臂。位置的新物体(即,右Vs的。左臂)必须来平衡在小鼠之间,以避免任何可能的混杂提示在房间中导致偏爱迷宫的一只手臂。
如上所述,将动物放在起点上。
与之前的会议(栖息地和训练会议)一样,观察是通过连接到记录系统的闭路摄像机完成的,以避免对动物造成不必要的压力或不受控制的提示(参见视频1 )。
 


D:\ Reformatting \ 2020-4-7 \ 1902471--1424 Giovanni Marsicano 652762 \ video 1.jpg


V IDEO 1. 代表的视频显示对象的探索迷宫的双臂


 


使用两个秒表并分配每个秒表来记录每个对象的探索时间,实验人员在不了解实验条件的情况下,测量每个对象的探索时间,以分析闭路摄像机获得的图像。记录目的以及一般活动的视频跟踪(每条手臂所花费的时间,每条手臂中的项数和行进距离)。
测试结束后,将动物从迷宫中移出并放回笼中。用水清洗迷宫后,从另一只动物开始(返回步骤C3)。
会议结束后,导出实验数据并分析结果(请参阅数据分析)。
 


注意:如果在训练和/或测试期间的总探索时间非常短(<5-10 s),则不考虑将这些小鼠的结果用于实验,这可能是由于:


该物体不足以完成任务(即可能会给动物带来压力或焦虑)。验证该物体是否已正确清洁,因为它可能含有先前动物的气味,并重新评估该物体以了解是否存在内在偏好或厌恶。
该动物不探索迷宫,因为运动赤字或不探索迷宫由于药物治疗和/或遗传manipulation.If有任务的执行过程中明确的运动变化,从排除动物分析。
 


数据分析


 


物体探索的量化
在培训和测试期间,实验者不愿对实验设计进行评分,而对探索性时间进行手动评分。由于自动系统目前难以准确区分尾巴和头部并正确计算探索时间,因此首选手动评分。认识到基于深度学习的姿态估计方法(Mathis 等,2018 ; Nath 等,2019)可能会提供一种可靠的方式来在不久的将来自动实现勘探量化。


在以下情况下对物体进行探伤:动物的鼻子正对着物体(< (2 Cm)如Robin所描述。Et Al ,2018 ; Oliveira Da Cruz。Et Al ,2019)。当物体:动物不探伤不会与对象互动,或者它位于对象顶部而没有面向对象。


注意:如果在整个会话过程中动物没有与两个对象至少互动一次,则该会话无效。


 


对象探索分析
当比较两个或两个以上实验组时,为了评估此探索过程中的差异,可以进行2种类型的一般分析:1)辨别指数(DI); 2)熟悉的Vs 。。另外,进行了全面的探索还表示了时间,以控制实验组之间总体勘探的可变性.DI的计算公式如下:


 


DI = [ 时间新颖- 时间熟悉)] / [(时间熟悉)+ (时间新颖)]


 


可以测量其他变量:每个手臂花费的时间,每个手臂中的项数和行进距离。 ±。SEM 双向小号Tudent'S 牛逼- 测试 。和单向ANOVA统计分析是统计测试中使用这些实验的比较差异有统计学意义时,显着性水平低于0.05。


 


预期成绩
在此协议中报告的实验条件下,在典型的训练过程中,每个对象的一般探索时间约为15-40 s。如果小鼠区分熟悉对象和新颖对象,则测试过程中的探索时间为对于熟悉的物体大约10-35 s,对于新物体大约30-60 s (图3和表1)。如果识别记忆力受损,如我们在注射四氢大麻酚(THC)的小鼠中所述(Puighermanal et al。(2009年),小鼠将在测试期间(20-40 s)探索相同时间的新颖物体和熟悉物体,因此它们不会区分它们(图3和表1)。


 


D:\ Reformatting \ 2020-4-7 \ 1902471--1424 Giovanni Marsicano 652762 \ Figs jpg \ Fig 3-word.jpg


图3的NOR任务一个例子的代表性曲线图。进一步的细节在表1 。


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                                                                 


 


1,一种实施例的表的预期数据和分析。ř 具有代表性的例子的Ñ 实验使用作为一个方便失忆剂量Delta9四氢大麻酚(THC,10毫克/千克,叶,光色)在CD1和C57BL / 6J小鼠,或其载体(VEH ,暗颜色的探索时间在训练会话(F')在第二对象上;)作为对照,训练session.The勘探时间(秒)在熟悉的物体(F)之后施用勘探在新对象中的时间(N);总探索时间(ET = F + F'或ET = F + N);在训练和测试过程中分配给每个对象的探索时间的百分比(%ET =( F / ET] x 10 0;%ET = [F'/ ET] x 100;%ET = [N / ET] x 100);在测试阶段计算歧视指数(DI = [N – F] / [N + F])。


CD1小鼠


C57BL / 6J


 


训练


测试


 


训练


测试


老鼠


F


F'


ET


F


ñ


ET


DI


老鼠


F


F'


ET


F


ñ


ET


DI


1个


42


45


87


35


67


102


0 。31


1个


55


45


100


18岁


49


67


0 。46


2


54


36


90


19


41


60


0 。37


2


27


二十三


五十


二十二


70


92


0 。52


3


39


34


73


12


39


51


0 。53


3


16


18岁


34


12


43


55


0 。56


四个


40


36


76


18岁


52


70


0 。49


四个


二十一


二十四


45


13


40


53


0 。51





53


58


111


26


45


71


0 。27





二十五


二十四


49


15


47


62


0 。52


6


43


49


92


19


72


91


0 。58


6


29


33


62


二十一


64


85


0 。51


7


61


56


117





47


57


0 。65


7


44


42


86


17


37


54


0 。37


意思


47 。4


44 。9


92 。3


19 。9


51 。9


71 。8


0 。46


意思


31


29 。8


60 。8


16 。9


五十


66 。9


0 。49


%ET


51 。4


48 。6


--


27 。7


72 。3


--


--


%ET


51


49


--


25 。2


74 。8


--


--


老鼠


F


F'


ET


F


ñ


ET


DI


老鼠


F


F'


ET


F


ñ


ET


DI


1个


37


32


69


二十五


32


57


0 。12


1个


28


32


60


27


39


66


0 。18


2


39


46


85


二十二


33


55


0 。2


2


34


28


62


26


二十二


48


-0 。10


3


30


38


68


27


二十四


51


-0 。06


3


29


45


74


33


34


67


0 。01


四个


51


52


103


54


59


113


0 。04


四个


35


28


63


二十一


二十三


44


0 。04





49


五十


99


37


45


82


0 。1





39


46


85


26


31


57


0 。09


6


57


42


99


19


16


35


-0 。09


6


45


51


96


29


31


60


0 。03


7


43


51


94


12


14


26


0 。08


7


29


38


67


41


38


79


-0 。04


意思


43 。7


44 。4


88 。1


28


31 。9


59 。9


0 。06


意思


34 。1


38 。3


72 。4


29


31 。1


60 。1


0 。03


%ET


49 。6


50 。4


--


46 。8


53 。2


--


--


%ET


47 。1


52 。9


--


48 。3


51 。7


--


--


 


 


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                


笔记


 


在设定行为任务的实验室中放置至少5天的小鼠。通常在锯末环境中将动物集体饲养(每笼3-6只动物),并用嵌套材料(棉立方),水/自由进食和控制温度(21±1℃)和湿度(55±10%)照明是在12小时黑暗/光照MAINTAINED:循环(上。在7 :00 一个。米。和关闭7 :00 在p ,中号。)和实验发生在开始的光照阶段(通常为9 :00 一。中号。-2 :00 的P- 。中号。)首页笼不应该改变,和动物不应该受到干扰时。住房设施应位于测试室附近,因为压力和运输的新颖性可能会改变勘探的方向。
选择合适的对象是此任务中必不可少的步骤,至少需要两对不同的对象,在选择对象时应仔细考虑诸如纹理(石头,木材,塑料,金属)(图4),颜色色调和形状等特征。要在这项任务中使用,重要的是要验证动物在训练过程中与每个对象互动所花费的时间是否相近。应避免对对象的任何偏爱或厌恶,因为这可能代表解释的重要偏见。为了避免这种可能的偏差,必须对动物同时进行两个对象的初步实验,并分析每个对象的探索时间,探索每个对象的时间应相似(约占总探索时间的50%首次向老鼠展示两个物体时,每个物体的探索时间会受到形状和物理特性的影响 但是,对象之间的差异必须足够大(颜色色调,纹理,形状),以便于在测试过程中区分两个对象。
 


D:\ Reformatting \ 2020-4-7 \ 1902471--1424 Giovanni Marsicano 652762 \ Figs jpg \ Fig 4.jpg


4.图对象使用在我们的NOR范式。在此图像中,你可以找到不同的例子对象使用在我们的NOR范式。你可以看到,不同的颜色,形状和材料都可以使用。


 


避免气味提示很重要,因此必须清洁动物之间,尤其是使用不同实验条件的实验之间的迷宫和物体。
对照动物可能不会表现出对新对象的适当区分。这可能是由于对一个对象的特定偏爱,也可能是由于训练和测试之间的不同,不受控制的条件(例如动物)引起的。在训练和测试之间的期间被动物看护者打扰了)或其他小鼠留下的嗅觉提示,并且在动物和/或训练之间没有很好地清洁它们。
极少数情况下,鼠标会跳出迷宫。为防止这些情况,可在迷宫上方放置透明的塑料盖。
 


致谢


 


我们感谢德尔菲娜·冈萨雷斯,纳塔莉Aubailly ,和所有的p 的的动物设施的ersonnel NeuroCentre 马让迪鼠标护理以及距离Parc de人员Recerca 生物医药巴塞罗那(PRBB)。我们也想感谢的人谁已经使用该task.This工作进行的实验工作是支持的INSERM(通用汽车),EU-FP7(FP7-PEOPLE-2013-IEF-623638到AB-G。),法国国家/ 法新社国立德拉RECHERCHE(ANR- 10-IDEX-03-02(AB-G),以及MINECO从AEI(RYC-2017-21776)到AB-G。


 


利益争夺


 


作者宣称没有利益冲突。


 


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引用:Oliveira da Cruz, J., Gomis-Gonzalez, M., Maldonado, R., Marsicano, G., Ozaita, A. and Busquets-Garcia, A. (2020). An Alternative Maze to Assess Novel Object Recognition in Mice. Bio-protocol 10(12): e3651. DOI: 10.21769/BioProtoc.3651.
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