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Delayed Spatial Win-shift Test on Radial Arm Maze
采用旋臂迷宫的延迟空间Win-shift试验   

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Abstract

The radial arm maze (RAM) is used to assess reference and working memory in rodents. This task relies on the rodent’s ability to orientate itself in the maze using extra-maze visual cues. This test can be used to investigate whether a rodent’s cognition is improved or impaired under a variety of experimental conditions. Here, we describe one way to test spatial working and reference memory. This delayed spatial win-shift (DSWS) procedure on the RAM was adapted from Packard and White (1990). The win-shift component of the test refers to the alternation of baiting, or rewarding, arms during the trial and test phase. The rodent is required to hold spatial information both within the task and across a delay to obtain the food-pellet reward (Taylor et al., 2003b). This task measures the incidence and type of memory errors made by the rodent both in the training and test phases of the learning task. A working memory error (re-entry of an arm that has been baited) can occur in both phases of the task, whilst a reference memory error (entry into an arm that has been baited during the training phase and is no longer baited) can only occur during the test phase.

Keywords: Delayed spatial win-shift (DSWS)(延迟空间win-shift (DSWS)), Radial arm maze (RAM)(旋臂迷宫(RAM)), Spatial working memory(空间工作记忆), Spatial reference memory(空间参考记忆), Trial phase(试验阶段), Test phase(测试阶段)

Background

The radial arm maze (RAM) can be used to examine the effects of hippocampal and prelimbic cortex (PLC) damage, ageing, as well as a variety of pharmacological agents (Wenk, 2001; Taylor et al., 2003b; Floresco et al., 1997; Vann et al., 2003). The hippocampus is widely accepted to be involved in both spatial working and reference memory. Lesions to the hippocampus in rodents have shown impairments in the ability to perform memory tasks, including the RAM, involving spatial navigation (O'Keefe and Nadel, 1978; Morris et al., 1982). The PLC region of the rat prefrontal cortex, the approximate equivalent of primate dorsolateral region of the prefrontal cortex (Groenewegen, 1988), is also involved in spatial working memory (Robbins, 1990). Taylor et al. have demonstrated that rodents with lesions to the PLC make more spatial reference and memory errors compared to controls in the delayed spatial win-shift (DSWS) procedure on the radial maze (Taylor et al., 2003b). The traditional RAM studies an animal’s explorative behaviour during the task, particularly investigating working memory (Seamans et al., 1995). The adaptation of the task to include the DSWS element is a well-established procedure in the literature. This technique investigates the rats’ ability to retain spatial information both within the task and across a delay (Taylor et al., 2003b; Lapish et al., 2008; De Luca et al., 2016).

Materials and Reagents

  1. Paper towels for cleaning the maze with ethanol
  2. Rats: Our experiments were conducted using 10-week old male Wistar rats but other rodents can be used. If using females, estrous cycle stage should be taken into consideration since spatial reference memory is attenuated during the pro-estrous phase of the cycle (Bowman et al., 2001; Pompili et al., 2010). If using young rats or adult mice, a mouse RAM of smaller dimensions should be used, see below.
    Notes:
    1. The rats are housed under normal controlled laboratory conditions, in weight-matched pairs, under a 12 h dark/light cycle, with ad libitum access to food and tap water prior to the task. The testing protocol should be undertaken during the light phase of the 12 h light cycle between 0700 and 1900 h. Circadian rhythm does not need to be accounted for throughout the experiment as the rodents undergo bi-daily sessions across a large part of the 12 h light phase. To ensure there are no time biases between the groups, spread the testing of control and treated rodents throughout the day. 
    2. During the testing protocol, access to food should be restricted to 80% of the rat’s usual food intake to encourage food-seeking behaviour in the maze. Adult (10-week old) male Wistar rats have an approximate daily food intake between 15-20 g during the 12 h dark phase and around 7 g during the 12 h light phase. Adult female Wistar rats have an approximate daily food intake between 10-15 g during the 12 h dark phase and around 5 g during the 12 h light phase (Stefanidis and Spencer, 2012).
    3. Prior to commencement and throughout the experiment, normal controlled laboratory light settings should be in place. The maximum allowable light intensity is equivalent to 300 lux at one meter height. There is no additional light source during the experimental protocol to alter the locomotor activity of the rodent.  
  3. Ethanol, 70% (v/v), diluted in distilled water
  4. Standard chow grain pellets (45 mg) (Bio-Serv, USA)

Equipment

  1. RAM
    The task is carried out in an eight-arm radial maze (Lafayette Instrument Company, USA), consisting of an octagonal central platform (34 cm diameter) and eight equally-spaced radial arms (87 cm long, 10 cm wide). At the end of each arm is a food well (2 cm in diameter and 0.5 cm deep; Figure 1). At the entrance to each arm is a clear Perspex door that controlled access in and out of the central area. Each door is controlled by a computerized control system (Lafayette) enabling the experimenter to regulate access to the arms. Salient visual cues of different geometric shapes and contrasting colours are placed around the maze on the walls of the room.
    Note: If using young rats or adult mice, a mouse RAM of smaller dimensions should be used. The central platform should be approximately 22 cm in diameter with arms 25 cm long, 6 cm wide and 6 cm high that is transparent to enable the mice to see extra-maze visual cues (Crusio and Schwegler, 2005). The mouse RAM, should be placed on the floor to avoid elevation-induced anxiety (Crusio and Schwegler, 2005).
  2. Digital video camcorder. In this experiment a Canon Legria FS200 was used. However, any camcorder can be used.
  3. Tripod. The video camcorder is attached to a tripod or to the ceiling to allow recording of the entire maze.


    Figure 1. Radial Arm Maze (Lafayette Instrument Company) with automated Perspex doors and spatial cues placed around the maze. The camcorder is placed on the tripod facing downwards (approximately 45° to the maze). The camcorder can also be attached to the ceiling directly above the maze.

Procedure

  1. 2-day habituation
    1. During habituation, all eight arms are opened and the rodents are placed in the center arena facing away from the investigator (to give consistent introduction to the spatial cues) and allowed to freely explore the maze. Three standard chow grain pellets (45 mg) are placed along each arm to encourage exploration. Rodents are habituated to the maze for two consecutive days, with two 10 min sessions 4 h apart (Figure 2).
    2. After the final habituation session, the rodents are returned to their home cages and provided with approximately 20 grains of standard chow food pellets per animal.
    3. The arena is cleaned with 70% ethanol after each animal (and session) and allowed to dry thoroughly before the next rat is tested.
    Note: Spatial cues such as colourful flags that are significantly different in appearance should be placed around the maze to allow the rat to orientate itself to its environment as albino rats are able to discriminate colour of certain wavelengths (Walton, 1933).


    Figure 2. Delayed win-shift radial arm maze schematic representation with spatial cues placed around the maze (spatial cues in this figure are for illustrative purposes and were not the actual cues used). During the training phase, rodents are allowed to explore four pseudo-randomly selected arms baited with grain pellets whilst the other four arms are blocked. After a 5 min inter-trial-interval spent in their home cages, rodents are placed back into the maze with all arms accessible for the test phase. Previously blocked arms are then baited with grain pellets.

  2. 13-day (26 trials) experimental phase
    Note: The duration of the experimental phase is dependent on the control group making a mean of no more than one error in the test phase. Therefore this experiment may go for less or longer than 13 days (26 trials). 
    1. At the completion of the habituation phase, rodents are placed back into their home cages until the bi-daily training and test phases the following day. On day three the rodents undergo bi-daily sessions consisting of a 5 min training phase, a 5 min inter-trial interval (ITI) where the rodent is returned to its home cage, and a 5 min test phase. The bi-daily sessions are 5 h apart (Figure 3).


      Figure 3. Timeline of the bi-daily habituation, training and test phases. A). Habituation sessions consist of two 10 min sessions with a 4 h interval. All eight arms are open and baited. B). Experimental bi-daily sessions. Each session includes a 5 min training phase, a 5 min inter-trial interval (ITI) and a 5 min test phase.

    2. Throughout the experiment, access to standard chow food can be restricted to approximately 80% of the rodents’ normal food intake as recorded at the commencement of the experiment to encourage explorative behaviour during testing (Halagappa et al., 2007). Body weight is measured three times a week to ensure rodents do not fall below 80% free-feeding weight. If this does occur the amount of daily food intake can be increased. An alternative strategy is also to impose food deprivation during the dark cycle (Olton and Schlosberg, 1978). 
    3. During the 5 min training phase, four of the eight arms are blocked. These are pseudo-randomly chosen (daily by the investigator) with no more than two adjacent arms being blocked at a time such that the combination of arms 1, 3, 4, 5 is excluded because 3, 4 and 5 are adjacent arms but 1, 2, 5, 7 is a valid combination because only 1 and 2 are adjacent (Floresco et al., 1997; Taylor et al., 2003b; Furgerson et al., 2014). The four unblocked arms are baited with three food pellets in each food well. During this phase of the experiment, the rodent is allowed to explore the maze for 5 min or until it has explored all of the open baited arms and retrieve the grain pellet rewards from all of them.
    4. The training phase can be video-recorded and scored using automated tracking programs such as EthovisionXT or can be manually scored in real time. An arm entry is recorded when the rodent moves all four paws off the central platform into the arm. Working errors (re-entry of an arm that had been baited and visited) can be recorded during this training phase.
    5. At completion of the training phase, rodents are returned to their home cage for 5 min. During this time, the maze is wiped down with 70% ethanol, allowed to dry completely and all eight arms opened. The previously blocked arms are baited with food pellets with the previously open arms left empty.
    6. After the ITI, rodents are placed back into the center of the maze and are allowed to explore all eight open arms for 5 min. This test phase can be video-recorded for automated scoring on camera or can be manually scored in real time. Two types of errors are recorded: working error (re-entry of an arm that had been baited and visited) and reference error (entry into an arm that had been baited during the training phase). The experiment is continued bi-daily until the control group makes a mean of no more than one error in the test phase (criterion), achieved after 26 trials in De Luca et al., 2016.
      Troubleshooting:
      1. If the rodent does not explore the maze during the habituation phase, a longer duration or additional sessions can be performed across the entire cohort before moving on to the test phase. The investigator may increase the amount of standard chow food pellets provided during the sessions as a greater incentive to explore.
      2. Rodents may attempt to jump from the arm they are exploring to an adjacent arm over the wall. To deter this from occurring the investigator should physically remove the rodent from the arm and place it back into the center of the maze facing away from the investigator.

Data analysis

  1. To analyse the DSWS RAM, the training and test phase data and the type of memory error, either spatial working or reference error, are analysed separately. The training phase can only investigate spatial working memory errors, while the test phase can investigate both spatial working and reference memory errors.
  2. The mean of the number of errors from four consecutive trials is taken and formed into bins (Brown and Giumetti, 2006). For example if the rats reached criterion after 14 days (or 28 trials), the analysis would include seven data points (with each data point containing 4 trials). Example data are given in Figure 4.
  3. The data are analysed using two-way analyses of variance (ANOVA)s with treatment and block as between factors, followed by Tukey’s post hoc tests as described (De Luca et al., 2016). We assume statistical significance when P ≤ 0.05.


    Figure 4. Example data showing incorrect arm entries during the test phase of the delayed win-shift radial arm maze. The numbers of incorrect entries from four consecutive test trials are binned together as a mean. The data are then analysed using two-way analyses of variance (ANOVA)s with treatment and block as between factors, followed by post hoc tests (De Luca et al., 2016).

Notes

  1. Statistical analysis can be performed on other measures using the DSWS procedure. The investigator can analyse the total number of errors made, the number of correct choices made before the first error, latency to reach the first arm from the central platform as well as the time it takes to complete the test (Taylor et al., 2003b).
  2. Spatial working and reference memory errors can often be described using other terms. Spatial working error (re-entry of an arm that had been baited and visited) is often referred to as within-phase errors and reference error (entry into an arm that had been baited during the training phase) as across-phase errors (Taylor et al., 2003b; Richter et al., 2013).

Acknowledgments

This behavioural procedure was adapted from previously published studies RAM (Packard and White, 1990; Taylor et al., 2003b; Taylor et al., 2003a; Floresco et al., 1997) and was performed by our group as described (De Luca et al., 2016). This work was supported by a Discovery Project Grant from the Australian Research Council (ARC) to SJ Spencer (DP130100508).

References

  1. Bowman, R. E., Zrull, M. C. and Luine, V. N. (2001). Chronic restraint stress enhances radial arm maze performance in female rats. Brain Res 904(2): 279-289.
  2. Brown, M. F. and Giumetti, G. W. (2006). Spatial pattern learning in the radial arm maze. Learn Behav 34(1): 102-108.
  3. Crusio, W. E. and Schwegler, H. (2005). Learning spatial orientation tasks in the radial-maze and structural variation in the hippocampus in inbred mice. Behav Brain Funct 1(1): 3.
  4. De Luca, S. N., Ziko, I., Sominsky, L., Nguyen, J. C., Dinan, T., Miller, A. A., Jenkins, T. A. and Spencer, S. J. (2016). Early life overfeeding impairs spatial memory performance by reducing microglial sensitivity to learning. J Neuroinflammation 13(1): 112.
  5. Floresco, S. B., Seamans, J. K. and Phillips, A. G. (1997). Selective roles for hippocampal, prefrontal cortical, and ventral striatal circuits in radial-arm maze tasks with or without a delay. J Neurosci 17(5): 1880-1890.
  6. Furgerson, M., Clark, J. K., Crystal, J. D., Wagner, J. J., Fechheimer, M. and Furukawa, R. (2014). Hirano body expression impairs spatial working memory in a novel mouse model. Acta Neuropathol Commun 2: 131.
  7. Groenewegen, H. J. (1988). Organization of the afferent connections of the mediodorsal thalamic nucleus in the rat, related to the mediodorsal-prefrontal topography. Neuroscience 24(2): 379-431.
  8. Halagappa, V. K., Guo, Z., Pearson, M., Matsuoka, Y., Cutler, R. G., Laferla, F. M. and Mattson, M. P. (2007). Intermittent fasting and caloric restriction ameliorate age-related behavioral deficits in the triple-transgenic mouse model of Alzheimer's disease. Neurobiol Dis 26(1): 212-220.
  9. Lapish, C. C., Durstewitz, D., Chandler, L. J. and Seamans, J. K. (2008). Successful choice behavior is associated with distinct and coherent network states in anterior cingulate cortex. Proc Natl Acad Sci U S A 105(33): 11963-11968.
  10. Morris, R. G., Garrud, P., Rawlins, J. N. and O'Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature 297(5868): 681-683.
  11. O'Keefe, J. and Nadel, L. (1978). The Hippocampus as a Cognitive Map. Clarendon Press.
  12. Olton, D. S. and Schlosberg, P. (1978). Food-searching strategies in young rats: Win-shift predominates over win-stay. J Comp Physiol Psychol 92(4): 609.
  13. Packard, M. G. and White, N. M. (1990). Lesions of the caudate nucleus selectively impair "reference memory" acquisition in the radial maze. Behav Neural Biol 53(1): 39-50.
  14. Pompili, A., Tomaz, C., Arnone, B., Tavares, M. C. and Gasbarri, A. (2010). Working and reference memory across the estrous cycle of rat: a long-term study in gonadally intact females. Behav Brain Res 213(1): 10-18.
  15. Richter, S. H., Zeuch, B., Lankisch, K., Gass, P., Durstewitz, D. and Vollmayr, B. (2013). Where have I been? Where should I go? Spatial working memory on a radial arm maze in a rat model of depression. PloS one 8(4): e62458.
  16. Robbins, T. W. (1990). The prefrontal cortex, 2nd edn. In J. M. Fuster (Ed.). International Journal of Geriatric Psychiatry. Raven, pp:348-348.
  17. Seamans, J. K., Floresco, S. B. and Phillips, A. G. (1995). Functional differences between the prelimbic and anterior cingulate regions of the rat prefrontal cortex. Behav Neurosci 109(6): 1063-1073.
  18. Stefanidis, A. and Spencer, S. J. (2012). Effects of neonatal overfeeding on juvenile and adult feeding and energy expenditure in the rat. PLoS One 7(12): e52130.
  19. Taylor, C. L., Latimer, M. P. and Winn, P. (2003a). Impaired delayed spatial win-shift behaviour on the eight arm radial maze following excitotoxic lesions of the medial prefrontal cortex in the rat. Behav Brain Res 147(1-2): 107-114.
  20. Taylor, C. L., Latimer, M. P. and Winn, P. (2003b). Impaired delayed spatial win-shift behaviour on the eight arm radial maze following excitotoxic lesions of the medial prefrontal cortex in the rat. Behav Brain Res 147(1-2): 107-114.
  21. Vann, S. D., Kristina Wilton, L. A., Muir, J. L. and Aggleton, J. P. (2003). Testing the importance of the caudal retrosplenial cortex for spatial memory in rats. Behav Brain Res 140(1-2): 107-118.
  22. Walton, W. E. (1933). Color vision and color preference in the albino rat. II. The experiments and results. J Comp Psychol 15(3): 373.
  23. Wenk, G. L. (2004). Assessment of spatial memory using the radial arm maze and Morris water maze. Current protocols in neuroscience 8-5.

简介

径向臂迷宫(RAM)用于评估啮齿类动物的参考和工作记忆。这个任务依赖于啮齿动物使用额外迷宫的视觉提示来定向自己在迷宫中的能力。该试验可用于研究啮齿动物的认知在多种实验条件下是否改善或受损。在这里,我们描述了一种测试空间工作和参考内存的方法。 RAM上的这种延迟的空间变化(DSWS)过程改编自Packard和White(1990)。测试的双赢组件是指在试验和测试阶段期间交替的诱饵或奖励武器。啮齿动物需要在任务内和延迟上保持空间信息以获得食物颗粒奖励(Taylor等人,2003b)。该任务测量啮齿动物在学习任务的训练阶段和测试阶段中发生的记忆错误的发生率和类型。在任务的两个阶段都可能发生工作记忆错误(已经被诱饵的手臂的重新进入),而参考记忆错误(进入在训练阶段被诱饵并且不再被诱饵的手臂)可以只发生在测试阶段。
关键字:延迟空间变换(DSWS),径向臂迷宫(RAM),空间工作记忆,空间参考记忆,测试阶段

[背景] 径向臂迷宫(RAM)可用于检查海马和初始皮质(PLC)损伤,衰老以及各种药理学药剂的影响(Wenk,2001; Taylor ,2003b; Floresco等人,1997; Vann等人,2003)。海马被广泛接受参与空间工作和参考记忆。在啮齿动物中对海马的损伤已经显示出执行记忆任务的能力的损伤,包括涉及空间导航的RAM(O'Keefe和Nadel,1978; Morris等人,1982)。大鼠前额叶皮质的PLC区域,大约相当于前额叶皮质的灵长类动物背外侧区域(Groenewegen,1988),也参与空间工作记忆(Robbins,1990)。 Taylor 。已证明与径向迷宫上的延迟空间win-shift(DSWS)程序中的对照相比,具有PLC损伤的啮齿动物产生更多的空间参考和记忆错误(Taylor等人,2003b)。传统的RAM研究动物在任务期间的探索行为,特别是研究工作记忆(Seamans等人,1995)。包括DSWS元素的任务的适配是文献中已经确立的过程。这种技术研究了大鼠在任务内和延迟上保留空间信息的能力(Taylor等人,2003b; Lapish等人,2008; De Luca等人, ,2016)。

关键字:延迟空间win-shift (DSWS), 旋臂迷宫(RAM), 空间工作记忆, 空间参考记忆, 试验阶段, 测试阶段

材料和试剂

  1. 用毛巾清洗迷宫的纸巾
  2. 大鼠:我们的实验使用10周龄的雄性Wistar大鼠进行,但可以使用其他啮齿动物。如果使用雌性,应考虑发情周期阶段,因为空间参考记忆在周期的发育期减弱(Bowman等人,2001; Pompili等人, ,2010)。如果使用年轻大鼠或成年小鼠,应使用较小尺寸的小鼠RAM,见下文。
    注意:
    1. 将大鼠置于正常受控的实验室条件下,以重量匹配的对,在12小时黑暗/光周期下,在任务之前随意获得食物和自来水。测试方案应在0700和1900小时之间的12小时光周期的轻相期间进行。在整个实验中不需要考虑昼夜节律,因为啮齿动物在12小时轻相的大部分期间经历每两天一次的会话。为确保各组之间没有时间偏差,在一整天内分散控制和治疗啮齿动物的测试。 
    2. 在测试方案期间,获得食物应当限于大鼠通常食物摄入的80%,以在迷宫中鼓励寻求食物的行为。成年(10周龄)雄性Wistar大鼠具有在12小时黑暗阶段期间15-20g之间的大约每日食物摄取和在12h光照阶段期间大约7g。成年雌性Wistar大鼠在12小时黑暗阶段期间的日摄食量约为10-15g,在12小时光照阶段期间约为5g(Stefanidis和Spencer,2012)。
    3. 在开始和整个实验之前,应当具有正常受控的实验室光设置。最大允许光强度相当于一米高处的300勒克司。在实验方案期间没有额外的光源来改变啮齿动物的运动行为。   
  3. 乙醇,70%(v/v),用蒸馏水稀释
  4. 标准食用颗粒(45mg)(Bio-Serv,美国)

设备

  1. RAM
    任务在八臂径向迷宫(Lafayette Instrument Company,USA)中进行,其由八边形中心平台(34cm直径)和八个等间隔的径向臂(87cm长,10cm宽)组成。在每个臂的末端是食物井(直径2cm,深0.5cm;图1)。在每个臂的入口是一个清晰的有机玻璃门,控制进出中央区域的进出。每个门由计算机控制系统(Lafayette)控制,使实验者能够调节手臂的进入。不同几何形状和对比颜色的显着的视觉提示被放置在房间墙壁上的迷宫周围。
    注意:如果使用幼鼠或成年小鼠,应使用较小尺寸的鼠标RAM。中心平台的直径大约为22厘米,臂长25厘米,宽6厘米,高6厘米,透明,使小鼠看到超迷宫的视觉线索(Crusio和Schwegler,2005)。鼠标RAM,应放置在地板上,以避免升高诱发的焦虑(Crusio和Schwegler,2005)。
  2. 数码摄像机。在本实验中,使用佳能Legria FS200。但是,可以使用任何摄像机。
  3. 鼎。视频摄像机连接到三脚架或天花板,以允许记录整个迷宫。


    图1.带有自动化Perspex门和放置在迷宫周围的空间线索的径向臂迷宫(Lafayette Instrument Company)。摄像机放置在面向下(与迷宫大约45°)的三脚架上。摄像机还可以直接连接到迷宫上方的天花板。

程序

  1. 2天习惯
    1. 在适应期间,所有八只手臂打开,啮齿动物被放置在面向远离研究者的中心竞技场(以一致地引入空间线索),并允许自由探索迷宫。沿每个臂放置三个标准食物颗粒(45mg)以促进勘探。啮齿动物习惯于迷宫连续两天,两个10分钟的过程相隔4小时(图2)。
    2. 在最后的习惯期后,啮齿动物返回其家笼,并且每只动物提供约20格令的标准食物颗粒。
    3. 在每只动物(和期间)后,用70%乙醇清洁球场,并在测试下一只大鼠之前使其充分干燥。
    注意:空间线索,例如外观上显着不同的多彩旗,应该放置在迷宫周围,以允许大鼠将自身定向到其环境,因为白化大鼠能够辨别某些波长的颜色(Walton,1933)。


    图2.延迟的双赢径向臂迷宫示意图,其中空间线索放置在迷宫周围(该图中的空间线索用于说明目的,而不是使用的实际线索)。在训练阶段,允许啮齿动物探索四个伪随机选择的用颗粒丸粒诱饵的臂,而其它四个臂被阻塞。在其家笼中花费5分钟的试验间隔后,将啮齿动物放回迷宫中,所有臂均可进入测试阶段。以前被封锁的武器然后用颗粒饵料
  2. 13天(26个试验)实验阶段
    注意:实验阶段的持续时间取决于对照组,在测试阶段中平均值不超过一个误差。因此,此实验可能持续时间少于或长于13天(26次试验)。 
    1. 在适应阶段完成后,将啮齿动物放回它们的家笼中,直到第二天的双日训练和测试阶段。在第三天,啮齿动物进行每日一次的会话,包括5分钟的训练阶段,5分钟试验间隔(ITI),其中啮齿动物返回到其笼中,和5分钟的试验阶段。每两天的会话间隔5小时(图3)。



      图3.每日习惯,训练和测试阶段的时间表。 A)。习惯会话包括两个10分钟的会话,间隔4小时。所有八个武器是开放和诱饵。 B)。实验双日会话。每个会话包括5分钟训练阶段,5分钟试验间隔(ITI)和5分钟试验阶段。

    2. 在整个实验中,标准食物的获得可以被限制为在实验开始时记录的啮齿动物的正常食物摄取的约80%,以在测试期间鼓励探索行为(Halagappa等人, 2007)。每周测量体重三次,以确保啮齿动物不低于80%的自由喂养重量。如果发生这种情况,可以增加每日食物摄入的量。另一种策略是在黑暗循环期间强加食物剥夺(Olton和Schlosberg,1978)。
    3. 在5分钟训练阶段期间,八个臂中的四个被封闭。这些是伪随机选择的(每天由研究者),其中一次阻塞不超过两个相邻的臂,使得排除臂1,3,4,5的组合,因为3,4和5是相邻的臂,但是1 ,2,5,7是有效的组合,因为只有1和2是相邻的(Floresco等人,1997; Taylor等人,2003b; Furgerson等人, et al 。,2014)。四个未阻塞的胳膊在每个食物井中有三个食物颗粒。在实验的这个阶段期间,允许啮齿动物探索迷宫5分钟或直到它已经探索了所有打开的诱饵臂,并且从所有它们中检索谷物颗粒奖励。
    4. 训练阶段可以使用诸如EthovisionXT的自动跟踪程序进行视频记录和评分,或者可以实时手动评分。当啮齿动物将所有四个爪从中心平台移动到臂中时,记录臂进入。在训练阶段,可以记录工作错误(已经被诱饵和被访问的手臂的重新进入)
    5. 在完成训练阶段,啮齿动物返回到他们的笼子5分钟。在此期间,用70%乙醇擦拭迷宫,使其完全干燥,并打开所有八个臂。先前封闭的手臂用食物颗粒填充,之前打开的手臂留空。
    6. 在ITI之后,将啮齿动物放回到迷宫的中心,并允许探索所有八个开放臂5分钟。该测试阶段可以被视频记录用于照相机上的自动记分或者可以实时手动记分。记录两种类型的错误:工作错误(已经被诱饵和被访问的手臂的重新进入)和参考错误(进入在训练阶段被诱饵的手臂)。实验持续每日两次,直到对照组在测试阶段(标准)中产生不超过一个误差的平均值,在De Luca等人2016年的26次试验后实现。
      疑难解答:
      1. 如果啮齿动物在适应阶段期间不探索迷宫,则可以在移动到测试阶段之前在整个队列中进行更长的持续时间或额外的会话。调查员可以增加会议期间提供的标准食品颗粒的数量,作为探索的更大动机。
      2. 啮齿动物可能试图从他们正在探索的手臂跳到墙上的相邻手臂。为了防止这种情况的发生,研究者应该将啮齿动物从手臂上除去,并将其放回到迷宫的背离调查者的中心。

数据分析

  1. 为了分析DSWS RAM,分别分析训练和测试阶段数据以及存储器错误的类型,空间工作或参考误差。训练阶段只能调查空间工作记忆错误,而测试阶段可以同时调查空间工作记忆和参考记忆错误。
  2. 取来自四个连续试验的误差数的平均值并形成为箱(Brown和Giumetti,2006)。例如,如果大鼠在14天(或28次试验)后达到标准,则分析将包括7个数据点(每个数据点包含4个试验)。示例数据如图4所示。
  3. 使用具有处理和阻断因素之间的双因素方差分析(ANOVA),接着如所描述的Tukey事后检验来分析数据(De Luca等人,2016)。当 P ≤0.05时,我们假设具有统计意义

    图4.示例数据显示在延迟的双赢径向臂迷宫的测试阶段期间不正确的臂条目。来自四个连续测试试验的不正确条目的数量被作为平均值分组在一起。然后使用具有处理和阻塞因素之间的双向方差分析(ANOVA),随后事后检验(De Luca等人,2016)来分析数据。

笔记

  1. 可以使用DSWS程序对其他测量进行统计分析。研究者可以分析所做错误的总数,在第一错误之前做出的正确选择的数量,从中央平台到达第一臂的等待时间以及完成测试所花费的时间(Taylor等人。,2003b)。
  2. 空间工作和参考存储器错误通常可以使用其他术语来描述。空间工作误差(已经被诱饵和被访问的手臂的再进入)通常被称为同相误差和参考误差(在训练阶段期间被引入到手臂中)作为跨相误差(Taylor ,2003b; Richter等人,2013)。

致谢

该行为过程改编自以前公开的研究RAM(Packard和White,1990; Taylor等人,2003b; Taylor等人,2003a; Floresco等人(De Luca等人,2016),通过我们的小组进行。这项工作是由澳大利亚研究委员会(ARC)发现项目拨款支持到SJ斯宾塞(DP130100508)。

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引用:De Luca, S. N., Sominsky, L. and Spencer, S. J. (2016). Delayed Spatial Win-shift Test on Radial Arm Maze. Bio-protocol 6(23): e2053. DOI: 10.21769/BioProtoc.2053.
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