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Jun 2019

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Delayed Alternation Task for the Study of Spatial Working and Long Term Memory in Rats
延迟交替任务用于研究大鼠空间工作记忆和长期记忆   

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Abstract

Memory systems can hold previously presented information for several seconds, bridging gaps between discontinuous events. It has been previously demonstrated that the hippocampus and the medial entorhinal cortex (mEC) are necessary for memory retention over delay intervals in alternation tasks. Here we describe the delayed alternation task, a spatial working memory (WM) task in which animals need to alternate between left and right sides of a figure-8 maze on a trial-by-trial basis to receive a reward. On each trial of this task, the rat has to remember the last episode and turn in the opposite direction compared to the previous trial. We manipulated the WM load by introducing delays of various lengths (10 s and 60 s) between trials. While other alternation task protocols use short delay intervals between trials, our protocol introduces a longer delay condition that requires animals to use long-term memory resources that are not necessarily supported by sequential neuronal firing patterns (i.e., time cells) as are seen with shorter delay intervals.

Keywords: Hippocampus (海马组织), Working memory (工作记忆), Rat (大鼠), Behavior (行为), Time cells (时间单元), Long-term memory (长期记忆), Delayed alternation task (延迟交替任务)

Background

A main function of the hippocampus and entorhinal cortex is to connect events separated by delay intervals (Eichenbaum, 2017; Robinson et al., 2017; Ainge et al., 2007; Sabariego et al., 2019). It has been proposed that retention of memory for these events is accomplished by cells that fire at successive moments in temporally structured experiences, known as time cells (Eichenbaum, 2017). However, while most studies have used alternation task protocols with short delay intervals that did not exceed 15 s (Ainge et al., 2007; Ito et al., 2015; Pastalkova et al., 2008), recent data suggest that time cell firing begins to disappear after approximately the first 20 s of the delay interval (Sabariego et al., 2019). Therefore, it is important to explore both behavioral performance and neuronal firing during delays that exceed these shorter time intervals. Consequently, the delayed alternation task protocol described here uses longer delays of 60 s, which require animals to use long term memory resources, likely due to a WM overload (Kim et al., 2013). This protocol represents a valuable tool for exploring the discrete serial firing patterns observed during shorter delay periods and the mechanisms involved in supporting WM and long-term memory maintenance sustained by other mechanisms, like local synaptic plasticity in hippocampus, sharp-wave ripples, or by activity patterns elsewhere in the brain. Moreover, and because it also includes a shorter delay condition (10 s delay), where sequential firing occurs, it provides an opportunity for the study of these discrete serial firing patterns observed during shorter delay periods.

Materials and Reagents

Note: All materials and reagents listed are examples based on what has been used successfully using the protocol provided; however, using different rat strains/sex/weights, and cereals could also be successful.

  1. Paper towels
  2. Chocolate sprinkles (i.e., Cocoa PebblesTM)
  3. Weigh boats (Chemglass, catalog number CLS-1815-002 )
  4. Pencil
  5. Eraser
  6. Behavioral sheets (see Appendix 1)
  7. Experimentally naive, male Long-Evans rats with weighing between 300 g-350 g (Charles Rivers Laboratories)
  8. Ethanol 70% (Fisher Scientific, Decon Laboratories, catalog number: 04-355-223 )

Equipment

  1. Figure-8-maze
    Dimensions: 101 cm long crosspiece connected to a center arm to the right and left arms at the top and bottom of the maze. Side pieces were 101 cm long as well. The top and bottom pieces were 150 cm long. All runways were 10 cm wide x 2 cm tall. The maze was elevated 50 cm from the ground (Figure 1). Weigh boats containing reward pieces (i.e., Cocoa PebblesTM) were attached underneath the reward locations and served as non visible odor cues to prevent subjects from using odor when taking their decision.


    Figure 1. The Figure-8-maze apparatus used for behavioral training and testing. A. Photography of the maze. Side barriers in the center stem indicate the delay site. Odor cues were provided in both sides of the maze, below the reward sites, in order to prevent a bias in choosing a particular arm (white weigh boats). B. Illustration of the maze indicating measurements of each section. C. Removable card-board barriers (hand crafted).

  2. Card-board barriers (hand crafted, dimensions: 21.6 cm x 28 cm)
  3. Stop-watch (Fisher Scientific, catalog number: ISO 17025 )
  4. Infrared camera (such as Acazon Conch-shape Infrared LEDs Security C amera, https://amzn.to/37XHKyG)
  5. Scale (Kent Scientific, catalog number SCL-1015 )
  6. Dim light source (i.e., desk lamp)

Software

Notes:

  1. Software listed below describes what has been used successfully using the protocol provided; however, using alternative software that allows for scoring of alternation could also be successful.
  2. For electrophysiological recordings a 4SX 64 channel Neuralynx system was used. We do not discuss electrophysiological procedures in this protocol but any high-density, low-noise electrophysiology system could be successful.
  1. Microsoft Excel
  2. Statistical package (i.e., SPSS, GraphPad prism, R)

Procedure

  1. General Notes
    Note: All behavioral sessions were performed during the dark phase (reversed light cycle: between 08:00 and 16:00) under dimly lit conditions. Performance of this task during the light phase could also be successful.
    1. Rats should be housed individually.
    2. Keep rats on a reversed 12 h light/dark cycle.
    3. Behavioral testing should take place during the dark cycle.
    4. Before behavioral training, rats should gradually be food deprived until reaching 85% of their ad libitum weight and they should be maintained at this weight throughout the experiment.
    5. Water should always be kept available.
    6. Weigh the rats daily, right before the behavioral session.
    7. Feed the rats in the end of the behavioral session.
    8. The order in which rats run the task should change daily so that the first rat running the task each day is different from the one that ran first the previous day.
    9. The experimenter/s should avoid any kind of unnecessary interference.
    10. Odor cues (food reward used as a reinforcer) should be provided to prevent the rat from making its decision based on the smell of the reward (Figure 1).
    11. Salient visual cues should be available in the room and they should be kept constant.
    12. Add some chocolate sprinkles (Cocoa PebblesTM or the cereal you will use as a reward) to the rats’ home-cages daily for 3 days before the habituation day to prevent neophobia.
    13. Handle the rats (~5 min each) daily for 3 days before the habituation day.

  2. Habituation
    1. On the fourth day, bring the rats (one by one) to the room where behavioral testing will take place and after weighting the rat, allow it to familiarize with the room and the apparatus by letting it freely explore the maze for 10 min with chocolate sprinkles spread all over the maze.
    2. Clean the maze with a 70% ethanol solution and paper towels at the end of the session and between animals. Make sure the maze is dry before the next rat is placed on the apparatus.
    3. Make sure the homecage bedding is kept clean to avoid rats from consuming their waste. This could interfere with the food deprivation procedure/weight loss.
    4. Feed all the rats at the end of the behavioral session according to their weights.
    5. Continue adding pieces of the food reward into the animal’s homecage to avoid neophobia.

  3. First Stage–Training
    1. On day five (or the fifth day), weigh the subject. Start placing the rat at the base of the center arm of the maze (the most distal point from the reward locations, see Figure 2).


      Figure 2. Illustration of the first stage of the training protocol. Each rat is placed at the base of the central stem of the apparatus, facing the choice arms (the asterisk notes the point where the animal is placed). Barriers are placed to force the rat to enter one of the choice arms. After it enters one of the arms, a reward will be delivered.

    2. Place a barrier to force the rat to enter one of the side arms where the reward (1 cocoa pebble) is located.
    3. After the rat consumes the reward, use the barriers to gently guide the rat to return to the base of the center arm. Then, switch the barrier at the far end of the central arm to force the rat to turn down the opposite choice arm for the next reward. It is crucial to not allow the rat to retrace its route at any point.
    4. Each session should last 20 min or 30 trials, whichever comes first.
    5. Repeat this procedure, alternating the path the rat can follow by using the barrier and the alternating arms until the rat is able to follow the pattern and run consistently, without trying to retrace its steps, during two consecutive days (criterion).
    6. Feed all the rats at the end of the behavioral session according to their weights.

  4. Second Stage–Training
    Note: Before this stage rats should have reached the criterion in stage 1.
    1. Weigh the subject.
    2. The use of barriers should be phased out as the rat is now allowed to enter either arm each time it reaches the end of the stem (see Figure 3).


      Figure 3. Illustration of the second stage of the training protocol. The use of barriers at the choice point should be phased out; each time the rat reaches the end of the stem it will be able to enter either arm, but it will be rewarded only for alternating arm entries in a “figure 8”-like pattern and they will be prevented from retracing their steps at any point.

    3. The first trial will be considered “trial 0” and the rat will always be rewarded.
    4. After this trial, rats will be rewarded only when running in alternating arm entries.
    5. Prevent the rats from retracing their steps at any point.
    6. This procedure should take 20 min or 30 trials (not including the initial run) to be accomplished, whichever comes first. If the animal is not improving its performance, increase the number of trials per day (up to 60).
    7. For each trial, the arm chosen will be scored as either correct (alternation) or incorrect (repeat entry into the previously chosen arm, see Appendix 1).
    8. Rats should be trained to a criterion performance of at least (90%) correct trials on 2 out of 3 consecutive days.
    9. Feed the animals at the end of the behavioral session according to their weights.

  5. Third Stage–Testing
    Note: Before this stage rats should have reached the criterion in stage 2. This third stage includes trials with delays.
    1. Weigh the subject.
    2. Rats should receive 30 trials daily, divided into 3 blocks of 10 trials each (no delay, 10-s delay and 60-s delay).
    3. The order of the trial blocks should be randomized every day. Depending on the questions the experimenters are trying to address, trial blocks can be doubled up to 60 so that each block is repeated, e.g., no delay, 10-s delay, 60-s delay, no delay, 10-s delay, 60-s delay). This structure would be beneficial for example if electrophysiological recordings are being performed simultaneously since a comparison between blocks of trials of the same type could be done.
    4. For delay trials, once the rat returns to the base of the stem, after the last trial of the previous block, confine the rat by placing two barriers at the base of the center stem (the delay site should be approximately 25 cm long). See Figure 4 and Video 1.


      Figure 4. Illustration of the third stage (testing). Delay testing will start once the rat has reached the criterion in stage 2. In each daily session, rats will receive 30 trials, grouped into three blocks of 10 trials. The order of the 3 blocks of delays will be pseudorandomized every day.

      Video 1. Example video of a 60-s delay trial during the third stage of behavioral testing. (All experimental procedures were approved by the Institutional Animal Care and Use Committees at the University of California, San Diego (protocol number S08276, 3-year approval since 4/19/2017).)

    5. Start a stop-watch to keep track of the delay duration.
      Note: The stop-watch should be in silent mode to avoid any auditory cues.
    6. At the end of the delay interval, remove the barrier that blocks the rat from accessing the central arm of the figure-8-maze, so that it can transverse freely into the stem and make its next choice.
    7. After the rat makes a choice and consumes the reward (if applicable), let it return to the delay zone on the center arm for the next trial to occur.
    8. Repeat this stage for at least 6 continuous days.
      Note: The duration of this stage can be variable depending on the question that the experimenters are trying to address.
    9. Clean the maze with a 70% ethanol solution and paper towels after running each rat. Make sure the maze is dry before the next rat is placed on it.
    10. Feed the animals at the end of the behavioral session according to their weights.

Data analysis

Note: Use Microsoft Excel or a similar software to record the behavioral data for each stage of the experiment.

  1. For the second stage enter data into an Excel spreadsheet with the following columns (see Figure 5):


    Figure 5. Excel spreadsheet showing the recorded data for the second stage and a summary table (bottom) of the results with the sum of the correct trials, incorrect trials, and the percentage of correct trials. Numbers 1, 2, 3 represent the rats. Columns with data recorded from rat 1 show an example of a rat meeting the criterion performance of 90 % correct trials on 2 out of 3 consecutive days, ready to move to the next stage.

    1. Number of trials.
    2. Day the experiment takes place (e.g., day 1, day 2, day 3).
    3. Separate the day sections into further columns with numbers that identify the rats (e.g., 1, 2, 3).
  2. For each trial the rat completes correctly enter 1, otherwise 0.
  3. At the end create a table that summarizes:
    The sum of the correct trials
    The sum of the incorrect trials
    The calculated percentage of correct trials
  4. For the third stage data follow the same instructions as for stage two with the addition of the randomized order of the delay blocks (no delay, 10 s, 60 s delays)
    Create a summary table of the correct, incorrect and percentage of the correct trials for each block (see Figure 6).


    Figure 6. Excel spreadsheet showing the recorded data for the third stage with the randomized delay orders, no delay (white), 10 s delay (light blue), 60 s delay (dark blue). Below is a summary of the correct, incorrect and percentage of the correct trials for each block of trials. Note that each day a the different block of trials (1st, 2nd and 3rd) will correspond to a different type of delay (no delay, 10 s delay and 60 s delay). Experimenters should consider this to organize their data before analysis. One option would be to enter the data always following a particular order (e.g., no delay, 10 s, 60 s delay) even if this is not the real order the animal follows that day.

  5. Use a statistical package (i.e., SPSS, GraphPad prism, R) to perform two-way anova inferences to analyze the differences between the groups of animals and the types of delays (see Figure 7 adapted from Sabariego et al. (2019) for a representative way to represent and analyze the data). Multiple comparisons of the significant interactions can be analyzed using a post hoc test like the Tukey’s test.


    Figure 7. Representative example of data plotting and analysis on a lesion experiment in the alternation task. Lesioned rats were impaired in the delayed versions but not in the continuous version of the task. The 7 days of testing were analyzed with a two-way ANOVAs (Group x Delay) that revealed main effects of Lesion and Delay and a Delay x Lesion interaction (P-value = 0.0001). Tukey’s post hoc tests: ^^^ P < 0.001 for control versus mEC lesion group comparison; *** P < 0.001 for control versus mEC+H lesion group comparison (lesions of mEC and hippocampus; ## P < 0.01 for mEC versus mEC+H lesion group comparisons (adapted from Sabariego et al., 2019).

Acknowledgments

This protocol was adapted from Sabariego et al. (2019). Recent work has been supported by Mount Holyoke College, Program of Neuroscience and Behavior.

Competing interests

The authors have no conflicts of interest.

Ethics

All experimental procedures were approved by the Institutional Animal Care and Use Committees at the University of California, San Diego (protocol number S08276, 3-year approval since 4/19/2017).

References

  1. Ainge, J. A., van der Meer, M. A., Langston, R. F. and Wood, E. R. (2007). Exploring the role of context-dependent hippocampal activity in spatial alternation behavior. Hippocampus 17(10): 988-1002.
  2. Eichenbaum, H. (2017). Time (and space) in the hippocampus. Curr Opin Behav Sci 17: 65-70.
  3. Ito, H. T., Zhang, S. J., Witter, M. P., Moser, E. I. and Moser, M. B. (2015). A prefrontal-thalamo-hippocampal circuit for goal-directed spatial navigation. Nature 522(7554): 50-55.
  4. Kim, S., Sapiurka, M., Clark, R. E. and Squire, L. R. (2013). Contrasting effects on path integration after hippocampal damage in humans and rats. Proc Natl Acad Sci U S A 110(12): 4732-4737.
  5. Pastalkova, E., Itskov, V., Amarasingham, A. and Buzsaki, G. (2008). Internally generated cell assembly sequences in the rat hippocampus. Science 321(5894): 1322-1327.
  6. Robinson, N. T. M., Priestley, J. B., Rueckemann, J. W., Garcia, A. D., Smeglin, V. A., Marino, F. A. and Eichenbaum, H. (2017). Medial entorhinal cortex selectively supports temporal coding by hippocampal neurons. Neuron 94(3): 677-688 e676.
  7. Sabariego, M., Schonwald, A., Boublil, B. L., Zimmerman, D. T., Ahmadi, S., Gonzalez, N., Leibold, C., Clark, R. E., Leutgeb, J. K. and Leutgeb, S. (2019). Time cells in the hippocampus are neither dependent on medial entorhinal cortex inputs nor necessary for spatial working memory. Neuron 102(6): 1235-1248 e1235.

简介

[摘要] 内存系统可以将先前显示的信息保留几秒钟,以弥合离散事件之间的差距。先前已经证明海马和内侧内嗅皮层(mEC)对于交替任务中延迟间隔的记忆保持是必需的。在这里,我们描述了延迟的交替任务,这是一种空间工作记忆(WM)任务,在该任务中,动物需要在逐个试验的基础上,在图8迷宫的左右两侧之间进行交替才能获得奖励。在执行此任务的每次试验中,大鼠都必须记住最后一集,并与先前的试验相比要朝相反的方向转动。我们通过在两次试验之间引入各种长度(10 s和60 s)的延迟来控制WM负载。而其他交替任务协议使用试验之间短的延迟时间间隔,我们的协议引入了要求动物使用的不一定是连续的神经元放电模式(支持长期记忆资源,较长的延迟情况,即,时间细胞)被认为与短延迟间隔。

[背景] 海马和内嗅皮层的主要功能是连接由延迟间隔分隔的事件(Eichenbaum,2017; Robinson 等,2017; Ainge 等,2007; Sabariego 等,2019)。有人提出,对于这些事件的记忆保留是通过在时间结构化体验中的连续时刻触发的细胞来实现的,这些细胞被称为时间细胞(Eichenbaum,2017)。然而,尽管大多数研究使用间隔时间不超过15秒的交替任务协议(Ainge 等人,2007; Ito 等人,2015; Pastalkova 等人,2008),但最近的数据表明,时空触发在延迟间隔的前20秒后开始消失(Sabariego et al。,2019)。因此,重要的是在超过这些较短时间间隔的延迟中探索行为表现和神经元放电。因此,此处描述的延迟交替任务协议使用60 s的较长延迟,这可能是由于WM超载而要求动物使用长期记忆资源(Kim 等人,2013)。该协议代表了一种宝贵的工具,可用于探索在较短延迟时间内观察到的离散连续触发模式,以及支持WM和由其他机制维持的长期记忆维持的机制,例如其他机制,例如海马的局部突触可塑性,尖波波纹或大脑其他部位的活动模式。此外,由于它还包括一个较短的延迟条件(10 s延迟),在该条件下会发生顺序点火,因此它为研究在较短的延迟时间内观察到的这些离散的连续点火模式提供了机会。

关键字:海马组织, 工作记忆, 大鼠, 行为, 时间单元, 长期记忆, 延迟交替任务

材料和试剂


 


注意:列出的所有材料和试剂都是基于使用提供的协议成功使用的实例。但是,使用不同的大鼠品系/性别/体重和谷物也可能成功。


纸巾
Cho 对照粉洒(即Cocoa Pebbles TM )
称重台(化学玻璃,目录号CLS-1815-002)
铅笔
橡皮
行为表(请参阅附录1)
实验性幼稚的雄性Long-Evans大鼠,体重在300克至350克之间(Charles Rivers Laboratories)
乙醇70%(Fisher Scientific,Decon Laboratories,目录号:04-355-223)
 


设备


 


图8-迷宫
尺寸:101厘米长的横档,连接到迷宫顶部和底部的左右臂的中心臂。侧件也长101厘米。顶部和底部的长度为150厘米。所有跑道均为10厘米宽x 2厘米高。迷宫距离地面50厘米(图1)。包含奖励件(即Cocoa Pebbles TM )的称重船被附在奖励位置下方,用作不可见的气味提示,以防止受试者在做出决定时使用气味。


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图1.jpg


                                                                                                                图1.用于行为训练和测试的图8迷宫装置。A. P 迷宫hotography。中央茎杆上的侧挡板表示延迟位置。在迷宫的两侧,奖励位置下方提供了气味提示,以防止选择特定手臂(白色称重船)时出现偏见。B. 我迷宫表示各部分的测量结果的llustration。C. 可移动的纸板挡板(手向后)。


纸板隔板(手工制作,尺寸:21.6 厘米x 28厘米)
秒表(Fisher Scientific,目录号:ISO 17025)
红外线照相机(如Acazon海螺形红外大号的ED股票C 相机,https://amzn.to/37XHKyG)                                                       
秤(Kent Scientific,目录号SCL-1015)
昏暗的光源(即台灯)
 


软件


 


笔记:


下列软件描述了已使用所提供的协议成功使用的软件;但是,使用允许对替代评分的替代软件也可能成功。
对于电生理记录,使用了4SX 64通道Neuralynx系统。我们不在此协议中讨论电生理程序,但是任何高密度,低噪声的电生理系统都可以成功。
微软Excel
统计包(即SPSS,GraphPad棱镜,R)
 


程序


 


A. 一般说明      


注意:所有行为环节均在昏暗条件下的黑暗阶段(逆光周期:08:00至16:00)进行。在轻型阶段执行此任务也可能会成功。


大鼠应单独饲养。
使大鼠保持相反的12小时明/暗周期。
行为测试应在黑暗周期中进行。
在进行行为训练之前,应逐渐剥夺大鼠的食物,直至达到其随意体重的85%,并且在整个实验过程中应保持体重不变。
应始终保持有水。
在行为训练之前,每天称重大鼠。
在行为训练结束时喂老鼠。
大鼠执行任务的顺序应每天更改,以便每天执行任务的第一只大鼠与前一天运行的第一只大鼠不同。
实验人员应避免任何不必要的干扰。
应提供气味提示(用作食物补强剂的食物奖励),以防止大鼠根据奖励的气味做出决定(图1)。
房间中应有明显的视觉提示,并且应保持恒定。
添加一些巧克力洒(可可卵石TM 的习惯一天,以防止恐新症前3天每天或你将作为奖励使用谷物),以老鼠的家笼。
在习惯日前3天每天处理大鼠(每只约5分钟)。
 


B. 习惯      


在第四天,将大鼠(一个一个地)带到进行行为测试的房间,并给大鼠称重后,让其自由地用巧克力探索迷宫10分钟,以使其熟悉房间和设备。洒满迷宫。
在实验结束时和动物之间,用70%乙醇溶液清洁迷宫,每条毛巾擦拭一遍。在将下一只老鼠放在设备上之前,请确保迷宫干燥。
确保家笼床上用品保持清洁以避免老鼠消耗其废物。这可能会干扰食物剥夺程序/体重减轻。
在行为结束时,根据体重给所有大鼠喂食。
继续将部分食物奖励添加到动物的笼子中,以避免恐惧症。
 


C. 第一阶段– 培训      


1. 在第五天(或第五天),称重对象。开始将大鼠放置在迷宫中心臂的底部(奖励位置最远的一端,见图2)。      


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图2.jpg


图2. 训练方案第一阶段的示意图。将每只大鼠放置在设备中央茎的底部,面对选择臂(星号表示放置动物的位置)。放置障碍物以迫使大鼠进入选择臂之一。进入其中一臂后,将获得奖励。


 


2. 放置障碍物以迫使大鼠进入奖励(1个可可卵石)所在的侧臂之一。      


3. 老鼠消耗完奖励后,使用障碍物轻轻地引导老鼠返回中心臂的底部。然后,在中心臂的远端切换障碍物,以迫使大鼠将对立的选择臂向下倾斜以获取下一个奖励。至关重要的是,不允许老鼠在任何时候追踪其路线。      


4. 每节应持续20分钟或30次试用,以先到者为准。      


5. 重复此过程,通过使用护栏和交替的手臂来改变大鼠可以跟随的模式,直到大鼠能够在连续两天(标准)内遵循模式并持续奔跑,而无需试图追回其步伐。      


6. 在行为训练结束时,根据体重为所有大鼠喂食。      


 


D. 第二阶段– 培训      


注意:在此阶段之前,大鼠应已达到阶段1的标准。


1. 称重主题。      


2. 应该逐步取消使用障碍物,因为现在允许大鼠每次到达茎的末端时都可以进入其中的任何一个臂(见图3)。      


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图3.jpg


图3. 训练方案第二阶段的示意图。在选择点上应避免使用障碍物;每次大鼠到达茎的末端时,它都可以进入任一手臂,但是只有以“图8”式的方式交替进入手臂时,它才会获得奖励,并且在任何时候都将阻止它们追踪步伐。


 


3. 第一个审判将被视为“审判0”,并且将始终奖励老鼠。      


4. 在该试验之后,只有在交替进入手臂时才对大鼠奖励。      


5. 防止老鼠在任何时候退步。      


6. 该过程应进行20分钟或30次试验(不包括初始运行),以先到者为准。如果动物没有改善其性能,请增加每天的试验次数(最多60次)。      


7. 对于每个试验,选择的手臂将被打分为正确(替代)或不正确(重复输入先前选择的手臂,请参见附录1)。      


8. 应训练大鼠在连续3天中的2天中至少执行(90%)正确试验的标准性能。      


9. 在行为训练结束时,根据动物的体重喂它们。      


 


E. 第三阶段– 测试      


注意:在此阶段之前,大鼠应已达到阶段2的标准。此第三阶段包括延迟试验。


1. 称重主题。      


2. 大鼠应每天接受30次试验,分为3组,每组10次试验(无延迟,延迟10秒和延迟60秒)。      


3. 试验区的顺序应每天随机化。根据实验者尝试解决的问题,可以将试验块加倍至60,以便重复每个块,例如,无延迟,10秒延迟,60秒延迟,无延迟,10秒延迟, 60秒延迟)。例如,如果同时进行电生理记录,则这种结构将是有益的,因为可以进行相同类型的试验块之间的比较。      


4. 对于延迟试验,一旦大鼠回到茎干的底部,则在前一个块的最后一次试验之后,通过在中心茎干的底部放置两个障碍物来限制大鼠(延迟部位应长约25 cm )。参见图4 和视频1 。      


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图4.jpg


图4.第三阶段的示意图(测试)。一旦大鼠达到第2阶段的标准,便会开始延迟测试。在每天的会议中,大鼠将接受30个试验,分为10个试验的三个组。每天将对3个延迟块的顺序进行伪随机处理。


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ video 1.jpg


V IDEO期间行为测试的第三阶段1.一个60-S延迟试验的实施例的视频


 


5. 启动秒表以跟踪延迟时间。      


注意:秒表应处于静音模式,以避免任何听觉提示。


6. 在延迟间隔结束时,移开阻挡老鼠进入8型迷宫中心臂的障碍,以便它可以自由地横穿茎部并做出下一步选择。      


7. 在大鼠做出选择并消耗了奖励(如果适用)之后,让它返回中心臂上的延迟区域,以进行下一次试验。      


8. 重复此阶段至少连续6天。      


注意:此阶段的持续时间可以根据实验者试图解决的问题而变化。


9. 每只大鼠跑动后,用70%乙醇溶液和纸巾清洁迷宫。在将下一只老鼠放在迷宫之前,请确保迷宫干燥。      


10. 在行为训练结束时,根据动物的体重喂它们。   


 


数据分析


 


注意:使用Microsoft Excel或类似软件记录实验每个阶段的行为数据。


1. 对于第二阶段,将数据输入到带有以下列的Excel电子表格中(请参见图5):      


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图5.jpg


图5. Excel电子表格显示了第二阶段的记录数据和结果汇总表(底部),其中包含正确试验,错误试验的总和以及正确试验的百分比。数字1、2、3代表大鼠。从大鼠1记录数据的列显示了一个示例,该示例在连续3天中有2天满足90%正确试验的标准性能,准备移至下一个阶段。


 


试用次数。
实验发生的日期(例如,第1天,第2天,第3天)。
将“天”部分分成可识别大鼠的数字(例如1、2、3)。
2. 对于每个试验,大鼠正确完成输入1,否则输入0。      


3. 最后,创建一个表,该表汇总:      


牛逼,他总结了正确的试验


牛逼,他总结了不正确的审判


牛逼正确的审判,他计算出百分比


4. 对于第三阶段,数据遵循与第二阶段相同的指令,并增加了延迟块的随机顺序(无延迟,10 s,60 s延迟)      


为每个块创建一个正确,不正确和正确试验百分比的摘要表(参见图6)。


 


D:\ Reformatting \ 2020-2-7 \ 1903021--1322 Marta Sabariego 795509 \ Figs jpg \图6.jpg


图6. Excel电子表格显示了第三阶段的记录数据,具有随机延迟顺序,无延迟(白色),10 s延迟(浅蓝色),60 s延迟(深蓝色)。以下是每个试验块的正确,不正确和正确试验百分比的摘要。请注意,每一天试验的不同的块(1 ST ,2 次和3 次)将对应于不同类型的延迟(无延迟,10秒延迟和60秒延迟)。实验人员在分析之前应考虑此点以整理其数据。一种选择是始终按照特定顺序(例如,无延迟,10 s,60 s延迟)输入数据,即使这不是当天动物遵循的真实顺序。


 


5. 使用统计软件包(即S PSS,G raph P ad棱镜,R)执行双向方差分析,以分析动物组之间的差异和延迟类型(参见图7,改编自Sabariego 等人) 。(2019)用于repres 表示和分析数据entative方式)。可以使用事后检验(例如Tukey检验)分析重大相互作用的多个比较。      


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-3-05 \ 3549--1903021--1322 Marta Sabariego 795509 \ Figs jpg \图7-updated.jpg


图7.交替任务中病变实验的数据绘图和分析的代表性示例。在延迟版本中受损的大鼠受损,但在连续版本中没有受损。用双向方差分析(组x延迟)分析测试的7天,该方差分析显示了病变和延迟的主要作用以及延迟x病变的相互作用(P 值= 0.0001)。Tukey的事后检验:^^^ P <0.001,用于对照组与mEC病变组的比较;*** P < 0.001对照与mEC的+ H病变组比较(LES mEC的和海马的离子; ## P <0.01相对于mEC的mEC的+ H病变组比较(一个从Sabariego dapted 。等人,2019) 。


 


致谢


 


该协议改编自Sabariego 等。(2019)。霍利奥克山学院的“神经科学与行为计划”支持了最近的工作。


 


利益争夺


 


作者没有利益冲突。


 


伦理


 


所有实验程序均已获得美国加利福尼亚大学圣地亚哥分校的机构动物护理和使用委员会批准(协议编号S08276,自2017年4月19日起3年批准)。


 


参考文献


 


JA的Ainge,MA的van der Meer,RF的Langston和ER的Wood(2007)。探索上下文相关的海马活动在空间交替行为中的作用。海马17(10):988-1002。
Eichenbaum,H.(2017年)。海马中的时间(和空间)。Curr Opin Behav Sci 17:65-70。
伊藤(HT),张(J.SJ),威特(Witter),议员,摩瑟(Moser),EI和摩瑟(Moser),MB(2015)。用于目标定向空间导航的前额-丘脑-海马回路。自然522(7554):50-55。
Kim,S.,Sapiurka,M.,Clark,RE和Squire,LR(2013)。人类和大鼠海马损伤后对路径整合的不同影响。美国国家科学院院刊110(12):4732-4737。
E. Pastalkova,V. Itskov,A. Amarasingham和G. Buzsaki(2008)。大鼠海马内部产生的细胞装配序列。科学321(5894):1322-1327。
NTM鲁滨逊,普利斯特利,JB,鲁克曼,JW,加西亚,AD,斯麦格林,弗吉尼亚州,马里诺,FA和Eichenbaum,H.(2017年)。内侧内嗅皮层选择性支持海马神经元的时间编码。神经元94(3):677-688 e676。
Sabariego,M.,Schonwald,A.,Boublil,BL,Zimmerman,DT,Ahmadi,S.,Gonzalez,N.,Leibold,C.,Clark,RE,Leutgeb,JK和Leutgeb,S.(2019)。海马中的时间细胞既不依赖于内侧内嗅皮质输入,也不依赖于空间工作记忆。Neuron 102(6):1235-1248 e1235。
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引用:Hoxha, M. and Sabariego, M. (2020). Delayed Alternation Task for the Study of Spatial Working and Long Term Memory in Rats. Bio-protocol 10(5): e3549. DOI: 10.21769/BioProtoc.3549.
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