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Sep 2019

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The Discrete Paired-trial Variable-delay T-maze Task to Assess Working Memory in Mice
利用T迷宫评价小鼠工作记忆的非连续配对变换延迟任务   

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Abstract

Working memory abnormalities involving the prefrontal cortex (PFC) dramatically contribute to poor functional outcomes in patients with schizophrenia and still represent an unmet therapeutic need. Studies in rodents might provide essential tools to understand the mechanisms underlying PFC-dependent working memory dysfunctions, as well as precious tools for genetic and pharmacological testing. However, proper tests assessing working memory and sensitive to PFC-dependent functions must be used. In this regard, the discrete paired-trial variable-delay T-maze task, equivalent to delayed non-match to sample tasks used in humans, has proved to be an effective paradigm to test PFC-dependent working memory dysfunctions with high predictive validity in human studies.

Keywords: Discrete paired-trial variable-delay T-maze (非连续配对变换延迟T迷宫), Working memory (工作记忆), Animal models (动物模型), Cognitive dysfunctions (认知障碍), Schizophrenia (精神分裂症)

Background

The term working memory refers to the type of memory that is active and relevant only for a short period of time, on the scale of seconds, while performing complex tasks such as reasoning, comprehension and learning. The concept of working memory evolved from that of short-term memory and now it stands at the interface between perceptual processes and long-term memory formation. The major components of working memory, as suggested by Baddeley's model (Baddeley, 2010), are: i) a short-term storage buffer for visual-spatial information that provides a virtual environment for physical simulation, calculation, visualization and optical memory recall (often referred as the visuo-spatial scratch pad); ii) a short-term storage buffer for verbal information (referred to as the phonological loop); (iii) a central executive component that is responsible for response selection and for coordinating the outputs of different short-term memory buffers; iv) an episodic buffer, in which complex multimodal events are integrated and stored online. In this model, the maintenance of specific information is governed by the buffer systems, while the regulation and coordination of this information (i.e., updating and maintenance of task goals, management of interference, and manipulation and transformations of stored content) are handled by the central executive processes. Impairments across the domains of phonological, visuo-spatial and central executive working memory are among the most consistently cognitive deficits observed in patients with schizophrenia (Castner et al., 2004; Forbes et al., 2009). The working memory central executive component has been associated in many studies with the function of the dorsolateral PFC. Instead, the storage buffers are thought to depend more on the inferior frontal cortex and parietal cortical areas (Wager and Smith, 2003). The remarkable correspondence between performances of human patients with frontal lobe lesions, PFC-lesioned monkeys and rodents and schizophrenic patients made the PFC-dependent tasks among the most used in behavioral/fMRI studies in schizophrenia and relative translational preclinical research (Callicott et al., 2000; Kellendonk et al., 2006; Papaleo et al., 2008; Barch et al., 2012).

There are numerous working memory tasks that have been employed and validated in rodents in order to reliably measure the maintenance of visuo-spatial information (Dudchenko, 2004; Kellendonk, et al., 2006; Papaleo et al., 2014), for example, the 8-arm radial maze “delayed non-match to sample” or ‘‘win-shift’’ (Seamans et al., 1995; Seamans and Phillips, 1994), the 8-arm maze “random foraging task” (Floresco et al., 1997; Seamans et al., 1995), the odor span tasks (Dudchenko, 2004; Young et al., 2007) and some paradigms of delayed matching and delay non-matching to sample position operant conditioning tasks (Dunnett, 1993). These tasks involve an initial “sample” or “forced run” phase in which the rodent is exposed to a visual target or an arm of the maze. Subsequently, in the “choice” phase that is run after a variable delay, the subject is simultaneously presented with the original sample (the “match”) and another visual target or arm (the “non-match”). These pairs of phases must be presented repeatedly but importantly, with randomly changing cues presented in the sample phase. Thus, the working memory construct is based on the fact that the tested rodent is required to integrate information held online (the sample phase) with the learned rule (non-match or match to sample). This paradigm has been mostly implemented in mice using T-mazes (Aultman and Moghaddam, 2001; Kellendonk et al., 2006). In this context, the discrete paired-trial variable-delay T-maze task seems to be similar to the human delayed response task and also it relies on mPFC functions (Kellendonk et al., 2006). Indeed, it is based on the delayed non-match to position paradigm where the delayed alternations responses are driven by food reinforcement (Ji et al., 2009; Leggio et al., 2019). In particular, during a ''sample'' or "forced run", the experimental subject is forced to explore an arm of the maze. Then, after a variable delay, in the ''choice run'' phase the subject has to choose between the original sample (the ''match'') and the opposite arm (the ''non-match''). Rodents are then presented with a sequence of randomly chosen forced runs, each followed by a choice run. The working memory construct is based on the fact that the experimental animal is required to integrate information held online (the forced run) with the learned rule (non-match or match to sample). In this context, the role of PFC for supporting complex executive functions is well acknowledged (Scheggia et al., 2018 and 2020), as well as the necessity of the hippocampus involvement for different tasks used to study the spatial working memory, such as the Radial Arm Maze (Myroshnychenko et al., 2017) and the Morris Water Maze (Morris et al., 1982; Dudchenko, 2004).

Materials and Reagents

  1. Male C57BL6/J mice (8-16 weeks old, Charles River Laboratories Italia, Italy)/Genetic mouse models
    Note: It is also possible to use female C57BL6/J mice (8-16 weeks old) after evaluation of estrous state as well as other mouse strains without any locomotor impairment.
  2. Food reinforcer (14 mg, 5TUL, TestDiet, Richmond, IN)
  3. Ethanol 10%

Equipment

  1. The T-maze apparatus
    The T-maze apparatus was built out of transparent Plexiglass (0.5 cm thick; dimensions of the alleys: 40 x 10.2 x 17.5 cm. Light levels will be: 20 ± 2 lux in the main alley; and 10 ± 2 lux in the side alleys). A recessed cup at the end of each side alley concealed the food reinforcement from view. In addition, care was taken to remove all visual cues that could be used by the animal to guide his choice: behavioral studies were carried out in a room without any visual landmarks or windows (Figure 1).


    Figure 1. T-maze apparatus. Photographs show the top view (A), front view (B) and side view (C) of the T-maze for mice used in Leggio et al. (2019).

Software

  1. GraphPad Prism software (GraphPad Software, USA) was used to perform the statistical analysis and artwork.

Procedure

In the discrete paired-trial variable delay T-maze task, mice were exposed to a sequence of randomly chosen forced runs, each followed by a choice run so that they were required to integrate information held online (the forced run) with the learned rule (non-match to sample) (Papaleo et al., 2012). After a week of single housing, body weight and 24-h food intake are recorded for 3 consecutive days. Animals are then food restricted throughout the experiment to maintain 90% of their ad libitum body weight. During the first week of food-restriction, each animal is also habituated to the food reinforcer (14 mg, 5TUL, TestDiet, Richmond, IN) for three consecutive days. Thereafter, animals are habituated to the T-maze apparatus allowing to retrieve the food reinforcement for 10 min/day for two consecutive days. After this, animals are exposed to 1 day of 10 forced-alternation runs. The animals are placed in the T-maze with one goal arm closed off and will have up to 2 min to locate and eat the food reinforcer in the open arm. After consuming the food pellet, each mouse is given an inter-trial period of at least 20 min in the home cage, and then placed back in the maze for another forced run. Beginning on the following day, the discrete-trial delayed alternation training starts. Following a randomly chosen forced run, and a 4-s delay interval in the home cage, the mouse is placed back in the maze with access to both arms. The food reinforcer is located in the opposite arm entered in the previous forced trial. After an inter-trial period of 20 min, the animal is placed back in the maze for another forced run-choice run paired trial, for a total of ten paired trials per day. A different pseudo-randomly chosen pattern of forced runs (e.g., R-R-L-R-L-L-RL-R-L) is used every day, but on a given day the same pattern is used for all animals. The apparatus must be cleaned with water and ethanol 10% after each trial with special attention to the choice point of the T-maze. Mice are trained at a 4-s inter-run delay and 20-min inter-trial delay for 20 days, or until the mouse successfully performs 8 correct out of 10 daily trials (80% choice accuracy) for 3 consecutive days. Animals that do not reach this criterion are eliminated. Once the mouse performs consistently at the 4-s inter-run delay, training at three additional inter-run delays (30, 60 and 240 s presented in a random fashion) and a 20-s inter-trial delay begins. Mice are given 4 trials of each delay on 4 consecutive days of testing for a total of 16 trials for each day.


Figure 2. Timeline of the discrete paired-trial variable-delay T-maze task. Training: each discrete trial consists of a forced run–choice run pair (inter-run delay of 4 s and inter-trial delay of 15-20 m); Criterion: 80% of correct choices in three consecutive days (10 paired-trial/day); Final phase: intra-trial delay of 4 s, 30 s, 60 s and 240 s and inter-trial delay of 20 s (16 paired-trial/day for four consecutive days) (Papaleo et al., 2012; Leggio et al., 2019).

Step by Step protocol

  1. Day 1: 5:00 PM: Weight and singly house each mouse. 1 min handling (H1). Change cage, water, filter and food.
  2. Day 2: Singly housing habituation.
  3. Day 3: Singly housing habituation. 1 min handling (H2).
  4. Day 4: Singly housing habituation.
  5. Day 5: Singly housing habituation. 1 min handling (H3).
  6. Day 6: Singly housing habituation.
  7. Day 7: Singly housing habituation.
  8. Day 8: Singly housing habituation.
  9. Day 9: About 5:00 PM weigh the food and each mouse. 
  10. Day 10: About 5:00 PM, weigh each mouse and food (24 h intake)
  11. Day 11: About 5:00 PM, weigh each mouse and food (24 h intake). Take all food off, leave water on. Change cage, filter and water.
  12. Day 12: 5:00 PM, Animals are partially food-deprived and remain that way throughout the experiment. Give ≈ 60% of their 24 h intake. Check nest status.
  13. Day 13: 5:00 PM. Give ≈ 60% of their 24 h intake. Arrive to 90% of their initial weight.
  14. Day 14: 5:00 PM. Give ≈ 60% of their 24 h intake. Arrive to 90% of their initial weight.
    Put also the rewarding food (about 10 pellets of 5TUL 14 mg).
  15. Day 15: 5:00 PM. Give 60% of their 24 h intake. Put also the rewarding food (about 10 pellets of 14 mg). Arrive to 90% of their initial weight.
  16. Day 16: 5:00 PM. Give 60% of their 24 h intake. Put also the rewarding food (10 pellets of 14 mg). Arrive to 90% of their initial weight.

Habituation period:
  1. Day 17: Each mouse is allowed to explore the maze with all doors raised for 10 min. Food is placed in both goal arms. Quantifying the time required to eat the 1st pellets. They have to eat all the food presented (max 16 pellets; 2 pellets in each goal arm).
  2. Day 18: Each mouse is allowed to explore the maze with all doors raised for 10 min. Food is placed in both goal arms. Quantifying the time required to eat the 1st pellets. They have to eat all the food presented (max 20 pellets; 2 pellets in each goal arm).
  3. Day 19: Animals are exposed to 1 day of 10 forced-alternation runs. Specifically, they are placed in the T-maze with one goal arm closed off and had up to 2 min to run and eat the reward in the open arm (only one pellet in the open arm). After consuming the reward, they are removed from the maze, one trial for each mouse. Run the experiment with about 10 cages in the room. Mice are tested back-to-back. One pellet at the end of the arms.
  4. Day 20-Day 39: Discrete paired-trial delayed alternation training
    1. Each discrete trial consists of a forced run–choice run pair.
    2. For the forced run, animals are constrained to enter a randomly chosen arm. After they consume the reward in that arm (timer started as soon as the animal has finished food) and a 4-s retention interval (this 4-s time is considered as a mean of time recorded to accomplished this procedure, see also Kellendonk et al., 2006), each mouse is placed back in the maze with access to both arms but with only the opposite arm entered in the previous forced run baited. On the forced run bait the alley with 1 pellet; on the free choice run bait the correct alley with 2 pellets. After an inter-trial period of about 15-20 min to avoid proactive interference from the last trial (Kellendonk et al., 2006), the mouse is placed back in the maze for another forced run. Test mice in the room back-to-back.
    3. A different, randomly chosen, pattern of forced runs (for example, R-R-L-R-L-L-R-L-R-L) is used every day. However, on a given day the same pattern is used for all animals. Animals are trained at a 4-s inter-run delay for 20 days, or until they successfully perform eight out of 10 trials (80% correct choices) for 3 consecutive days. Animals that don’t reach this criterion are rejected.
    4. Light cycle: 6-6. Habituation about 8:45 AM. Test from 9:45 AM to 5:00 PM Reefed animals about 5:00 PM. 
  5. Day 40 or earlier: Once an animal performs consistently at the 4-s inter-run delay, training at three additional delays (30, 60 and 240 s) and with an inter-trial delay of only 20 s began. Mice are tested at all 4 inter-run delays for 4 consecutive days with 16 paired-trial/day (4 paired trial at each delay every day).

Note: This procedure is considered stressful for animals because each mouse is consecutively tested in several trials (16-paired trials) for, at least, 45 min. Conversely, during the training phase of the paradigm, each mouse performs 10-paired trials per day with an inter-trial-interval of 10 min.

Data analysis

  1. All data generally assume a normal distribution and then they are subjected to parametric tests (one- or two-way analysis of variance (ANOVA) and two-way ANOVA with repeated measures when appropriate).
  2. The Grubbs test can be performed to identify outliers (Leggio et al., 2019, Supplementary Information).
  3. Latency to eat the 1st pellets (in seconds, Days 17-18) is analyzed typically via two-way repeated measures (RM) ANOVA to reveal main effects and interaction (e.g., genotype × day) followed by a suitable post-hoc test for pairwise comparisons such as the Newman-Keuls test. Days to reach criterion (in days, Days 20-39) during the training phase can be analyzed through an unpaired (two sample) t-test or a one-way ANOVA to reveal main effect (e.g., genotype or treatment) according to the number of experimental groups (two of more). Finally, percentage of correct choices (as index of working memory) during the final phase (day 40 or earlier) is also generally analyzed through two-way RM ANOVA to reveal main effects and interaction (e.g., genotype × delay), followed by an appropriate post-hoc test.
  4. Graphs in Figure 3 display Latency to eat (A), Days to criterion (B), and correct choices (%, C) that wild-type (WT) and knockout mice littermates (here Dys+/- mice) performed during the task (adapted from Leggio et al., 2019). Both groups learned similarly to run quickly into the maze to retrieve the reward, as indicated by the significant decrease of the latency to eat during the second day (Day 18) of exposure to the reward compared to the first day (Day 17). Moreover, both groups required the same number of days to reach the criterion of 80% of correct choices for 3 consecutive days. However, although both groups exhibited delay-dependent behavior (progressive increase of errors with longer delays), Dys+/- mice showed significant working memory deficits than WT mice at both 4 and 30 s intra-run delays.


    Figure 3. WT (n = 12) and Dys+/− (n = 10) mice were tested in the discrete paired-trial variable-delay T-maze task. A. Latency to retrieve the reward (Genotype F(1,20) = 4.982, P = 0.0372; Day F(1,20) = 68.04, P < 0.0001, Genotype × Day F(1,20) = 5.37, P = 0.031; Post hoc: **P < 0.01 vs. Day 17). B. Days needed to reach the criterion (P = 0.88). C. Percentage of correct choices with different randomly presented intra-run delays (4, 30, 60, and 240 s) and an inter-trial delay of 20 s (Genotype F(1,20) = 5.95, P = 0.0241; Delay F(3,60) = 14.93, P < 0.0001, Genotype × Delay F(3,60) = 0.65, P = 0.58; Post hoc: *P < 0.05 vs. each WT time-point). The values are the means ± S.E.M. (adapted from Leggio et al., 2019).

  5. Graphs in Figure 4 display the data distribution of C57Bl6/J WT mice (n = 46) obtained by clustering different experiments. Latency to eat (A), Days to criterion (B), and correct choices (%, C) that C57Bl6/J wild-type performed during the task (adapted from Papaleo et al., 2008 and 2014; Leggio et al., 2019).


    Figure 4. Data distribution of C57Bl6/J WT mice (n = 46) obtained by clustering different experiments. A. Latency to retrieve the reward (Paired t-test; ***P < 0.001). B. Days needed to reach the criterion. C. Percentage of correct choices with different randomly presented intra-run delays (4, 30, 60, and 240 s) and an inter-trial delay of 20 s (Delay F(3,180) = 22.49, P < 0.0001). The values are the means ± S.E.M. (adapted from Papaleo et al., 2008 and 2014; Leggio et al., 2019).

Notes

  1. Animals were food restricted throughout the experiment to maintain 90% of their ad libitum body weight. The maintenance of the 90% of mice’s initial weight is crucial. Mice that do not maintain this body weight condition tend to slow down or do not move into the maze.
  2. The ideal number of mice that a researcher should test in this behavioral task is 10-12. Testing more animals in the same experiment could be too challenging because of the long duration of the daily procedure, mostly in the last phase of the test (45-60 min per mouse).
  3. During the experiment, researchers should avoid to put the mouse on the grid of the cage. It is less stressful for the mouse picking up it from the home cage and placing it directly into the start position of the maze.

Acknowledgments

We thank Dr. M. Morini, D. Cantatore, R. Navone, G. Pruzzo, A. Parodi, A. Monteforte and C. Chiabrera for technical support. This work was supported by funding from the Istituto Italiano di Tecnologia, the University of Catania, the Brain and Behavior Research Foundation (2015 NARSAD 23234), and the Compagnia di San Paolo (2015-0321). The methodology described was previously used in Leggio et al. (2019).

Competing interests

The authors declare that they have no competing interests.

Ethics

Animal sample size was chosen based on studies using related methods and is similar to what is generally employed in the field. Randomisation was not used to assign animals to experimental groups, and the investigator was blinded to the genotype of animals. All experiments were carried out according to EU Directive 2010/63/EU and the Institutional Animal Care and Use Committees of both Catania University and the Istituto Italiano di Tecnologia (IIT).

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简介

[摘要] 涉及前额叶皮质(PFC)的工作记忆异常极大地导致了精神分裂症患者的功能转归不良,并且仍然代表着未满足的治疗需求。对啮齿动物的研究可能会提供必要的工具,以了解潜在的PFC依赖的工作记忆功能障碍的机制,以及进行遗传和药理学测试的宝贵工具。但是,必须使用评估工作记忆并对PFC相关功能敏感的适当测试。在此方面,已证明离散的配对试验可变延迟T迷宫任务等效于人类中使用的样本任务的延迟不匹配,已被证明是一种有效的范例,可以预测PFC相关的工作记忆功能障碍并具有较高的预测效度在人类研究中。

[背景] 术语工作记忆是指在执行诸如推理,理解和学习之类的复杂任务时,仅在短时间内处于活动状态且相关的记忆类型,以秒为单位。工作记忆的概念是从短期记忆的概念演变而来的,现在它处于感知过程和长期记忆形成之间的接口。正如Baddeley模型(Baddeley,2010年)所建议的那样,工作记忆的主要组成部分是:i)视觉空间信息的短期存储缓冲区,为物理模拟,计算,可视化和光学记忆召回提供虚拟环境(通常称为视觉空间便签本);ii)用于口头信息的短期存储缓冲区(称为语音循环);(iii)中央执行部分,负责选择响应并协调不同短期记忆缓冲区的输出;iv)情景缓冲器,其中集成了复杂的多模式事件并在线存储。在此模型中,特定信息的维护由缓冲系统控制,而此信息的调节和协调(即任务目标的更新和维护,干扰管理以及存储内容的操纵和转换)由维护人员负责。中央执行程序。语音,视觉空间和中枢执行工作记忆域的障碍是在精神分裂症患者中观察到的最一致的认知缺陷之一(Castner 等,2004; Forbes 等,2009)。在许多研究中,工作记忆中央执行器组件都与背外侧PFC的功能相关联。取而代之的是,认为存储缓冲液更多地取决于额下皮层和顶叶皮层区域(Wager和Smith,2003)。人类额叶病变患者,PFC损伤的猴子和啮齿动物以及精神分裂症患者的表现之间的显着对应关系使PFC依赖性任务成为精神分裂症的行为/ fMRI研究和相对转化的临床前研究中最常用的任务(Callicott 等, 2000; Kellendonk 等,2006; Papaleo 等,2008; Barch 等,2012)。

为了可靠地测量视觉空间信息的维护情况,在啮齿动物中已经采用并验证了许多工作记忆任务(Dudchenko,2004年;Kellendonk 等人,2006年; Papaleo 等人,2014年),例如, 8臂迷宫“延迟到样品不匹配”或“双赢” (Seamans 等,1995;Seamans 和Phillips,1994),8臂迷宫“随机觅食任务” (Floresco 等)等人,1997;Seamans 等人,1995),气味跨度任务(Dudchenko,2004;Science 等,1995)。 Young 等。,2007 )和延迟匹配和延迟不匹配到样本位置操作条件任务的一些范式(Dunnett,1993)。这些任务涉及初始的“样本”或“强制运行”阶段,在此阶段中,啮齿动物会暴露于视觉目标或迷宫的手臂。随后,在可变延迟后运行的“选择”阶段,对象同时被呈现原始样本(“匹配”)和另一个视觉目标或手臂(“不匹配”)。这些阶段对必须重复出现,但重要的是,在样本阶段中出现随机变化的提示。因此,工作记忆结构是基于以下事实:需要测试的啮齿动物将在线保存的信息(样本阶段)与学习的规则(不匹配或与样本匹配)进行整合。这种范例已在大多数使用T- mazes的小鼠中实现(Aultman 和Moghaddam,2001;Kellendonk 等,2006)。在这方面,所述离散的成对审可变延迟T迷宫任务似乎是类似于人类延迟响应任务并且还依赖于mPFC的功能(Kellendonk 等人,2006)。确实,这是基于延迟的不匹配位置范式,其中延迟的交替反应是由食物强化驱动的(Ji 等人,2009 ; Leggio 等人,2019)。特别地,在“样本”或“强制运行”期间,实验对象被迫探索迷宫的手臂。然后,经过可变的延迟后,在“选择运行”阶段,受试者必须在原始样本(“匹配”)和对立臂(“不匹配”)之间进行选择。然后为啮齿动物提供一系列随机选择的强制奔跑,每个奔跑之后都有一个选择奔跑。工作记忆的构建基于以下事实:需要实验动物将在线保存的信息(强制运行)与学习的规则(不匹配或与样品匹配)进行整合。 在这种情况下,PFC在支持复杂执行功能中的作用已广为人知(Scheggia et al。,2018 和2020),以及海马体参与研究空间工作记忆的不同任务的必要性,例如放射状迷宫(Myroshnychenko 等,2017)和莫里斯水迷宫(Morris 等,1982; Dudchenko ,2004)。

关键字:非连续配对变换延迟T迷宫, 工作记忆, 动物模型, 认知障碍, 精神分裂症

材料和试剂


 


1. C57BL6 / J雄性小鼠(8-16周大,意大利Charles River Laboratories,意大利)/遗传小鼠模型      


注意:我t是也可以使用动情状态以及其它小鼠品系的评估后雌性C57BL6 / J小鼠(8-16周龄)没有任何机车运动障碍。


2. 食品增强剂(14毫克,5TUL,TestDiet,里士满,印第安纳州)      


3. 乙醇10%      


 


设备


 


T型迷宫装置
T型迷宫装置由透明的有机玻璃(0.5厘米厚;小巷的尺寸:40 x 10.2 x 17.5厘米)制成。光照强度为:主小巷为20±2 lux,侧面为10±2 lux小巷)。每个侧巷子的末端都有一个凹入的杯子,看不见食物强化物。此外,要小心删除动物可以用来指导选择的所有视觉线索:在没有任何视觉标志或窗户的房间内进行行为研究(图1)。


 


D:\ Reformatting \ 2020-4-7 \ 1902963--1436 G.Leggio 828946 \ Figs jpg \ Fig 1.jpg


图1. T型迷宫装置。照片显示了Leggio 等人使用的T型迷宫的俯视图(A),正视图(B)和侧视图(C)。(2019)。


 


软件


 


使用GraphPad Prism软件(美国GraphPad软件)进行统计分析和绘制图稿。
 


程序


 


在离散配对试验的可变时延T迷宫任务中,将小鼠暴露于一系列随机选择的强制奔跑中,每只小鼠随后进行一次选择奔跑,以便要求它们将在线保存的信息(强制奔跑)与所学习的规则相结合。 (与样品不匹配)(Papaleo 等,201 2 )。单身居住一周后,连续3天记录体重和24小时食物摄入量。然后在整个实验期间限制动物的食物,以保持其随意体重的90%。在禁食的第一周,每只动物还要连续三天习惯使用食物强化剂(14 mg,5TUL,TestDiet,Richmond,IN)。此后,使动物习惯于T型迷宫设备,连续10天/天以10分钟/天的速度恢复食物。此后,将动物暴露于10次强制交替运行的1天。将动物放在T型迷宫中,一只守门员臂关闭,最多需要2分钟的时间才能在敞开的胳膊中找到并吃掉食物强化剂。食用食物颗粒后,将每只小鼠在家庭笼子中进行至少20分钟的审判,然后放回迷宫中进行另一次强制奔跑。从第二天开始,离散试验延迟交替训练开始。在随机选择的强制运行以及在家用笼中有4 s的延迟间隔后,将鼠标放回迷宫中,可以接触到两只手臂。食物强化剂位于上一次强制试验中输入的对侧臂中。试用期为20分钟后,将动物放回迷宫中进行另一项强制选择选择配对试验,每天总共进行十次配对试验。每天使用不同的伪随机选择强制运行模式(例如RRLRLL-RL-RL),但在给定的一天,所有动物都使用相同的模式。每次试验后,必须用水和10%的乙醇清洁设备,并特别注意T迷宫的选择点。对小鼠进行4 s的运行间隔延迟和20分钟的试验间隔延迟进行训练,持续20天,或者直到鼠标连续3天成功执行10次每日试验中的8次正确(80%选择准确性)。达到此标准的动物将被淘汰。一旦鼠标以4 s的运行间隔延迟一致地运行,就开始以三个附加的运行间隔延迟(以随机方式呈现30、60和240 s)进行训练,并开始20 s的试验间隔延迟。在连续的4天测试中,对小鼠进行4次每个延迟的试验,每天总共进行16次试验。 


 


D:\ Reformatting \ 2020-4-7 \ 1902963--1436 G.Leggio 828946 \ Figs jpg \ Figure。 2.jpg


图2.离散成对的可变延迟T型迷宫任务的时间线。培训:每个离散试验都包括一个强制选择跑对(两次间延迟4 s,一次间延迟15-20 m);评判标准:连续三天(每天10次配对试验)有80%的正确选择;最终阶段:审判内延迟4 s,30 s,60 s和240 s,审判间延迟20 s(连续四天每天16次配对/天)(Papaleo 等人,2012 ;Leggio 等人。,2019)。


 


分步协议


第1天:5 :00 PM :w ^ 八单独容纳每个鼠标。处理1分钟(H1)。更换笼子,水,过滤器和食物。
第2天:š ingly壳体习惯。
第3天:š ingly壳体习惯。处理1分钟(H2)。
第4天:š ingly壳体习惯。
第5天:š ingly壳体习惯。处理1分钟(H3)。
第6天:š ingly壳体习惯。
第7天:š ingly壳体习惯。
第8天:š ingly壳体习惯。
第9天:一个回合5:00 PM 权衡食品和每个鼠标。
第10天:甲回合5:00 PM ,称量每只小鼠和食(24小时摄入)
第11天:甲回合5:00 PM ,称量每只小鼠和食(24小时摄入量)。拿掉所有食物,放开水。更换笼子,过滤器和水。
第12天:5:00 PM ,动物是部分食品被剥夺,并保持整个实验的方式。摄入约其24小时摄入量的60%。检查嵌套状态。
第13天:5:00 PM 。摄入约其24小时摄入量的60%。达到其初始重量的90%。
第14天:5:00 PM 。摄入约其24小时摄入量的60%。达到其初始重量的90%。
还要放入有益的食物(约10粒5TUL 14毫克)。


15日:5:00 PM 。给予其24小时摄入量的60%。还要放入有益的食物(约10粒,每粒14毫克)。达到其初始重量的90%。
16日:5:00 PM 。给予其24小时摄入量的60%。还要放入有益的食物(10粒,每粒14毫克)。达到其初始重量的90%。   
 


居住期:


第17天:允许所有老鼠抬起所有门,探索迷宫10分钟。将食物放入两个目标臂中。量化吃1个需要的时间ST 颗粒。他们必须吃掉所有提供的食物(最多16粒;每个目标臂2粒)。
第18天:允许所有老鼠抬起所有门,探索迷宫10分钟。将食物放入两个目标臂中。量化吃1个需要的时间ST 颗粒。他们必须吃掉所有提供的食物(最多20粒;每个目标臂2粒)。
第19天:将动物暴露于10次强迫交替的第1天。具体来说,将它们放在T形迷宫中,一个门臂闭合,最多2分钟即可在开放臂中运行并吃掉奖励(开放臂中只有一个药丸)。消耗完奖励后,将它们从迷宫中移出,每只老鼠进行一次试验。在大约10个笼子里进行实验。小鼠背对背进行测试。手臂末端有一颗药丸。
天20- d 着y 39:离散配对试验推迟交替训练
每个离散试验均包含一个强制选择运行对。
对于强制奔跑,必须限制动物进入随机选择的手臂。当他们在那条手臂上消耗了奖励(动物吃完食物后立即开始计时)和4 s的保留间隔(这4 s的时间被视为完成此过程所记录的时间的平均值,另请参见Kellendonk 等)等人,2006),将每只小鼠放回迷宫中,可以接触到两只手臂,但在先前的强制诱饵中只有另一只手臂进入。在强行诱饵下,胡同中有1颗药丸;在自由选择的条件下,诱饵诱捕2粒正确的胡同。在大约15-20分钟的试验间隔后,以避免来自上次试验的主动干预(Kellendonk 等,2006),将小鼠放回迷宫中进行另一次强制性奔跑。在房间中连续测试小鼠。
每天使用不同的,随机选择的强制运行模式(例如RRLRLLRLRL)。但是,在给定的一天,所有动物都使用相同的模式。对动物进行20天的运行间隔延迟训练,持续20天,或者直到它们连续3天成功执行了十分之八的试验(正确选择的80%)。不符合此标准的动物将被拒绝。
光周期:6-6。习惯约为8 :45 AM 。从9测试:45 AM 〜5 :00 PM 补料动物约5 :00 PM。
第40天或更早:一旦动物在4 s 的跑步间隔内表现稳定,就开始以另外三个延迟(30、60和240 s)进行训练,而试验间的延迟仅为20 s。连续4天对小鼠进行所有4 次运行间延迟测试,连续16天每天进行16次配对试验(每天每次延迟4次配对试验)。
 


注意:由于对每只小鼠进行了至少45分钟的多次试验(16对配对试验),因此对动物造成压力。相反,在范式的训练阶段,每只小鼠每天进行10对配对试验,试验间隔为10分钟。


 


数据分析


 


所有数据通常都呈正态分布,然后接受参数测试(单向或双向方差分析(ANOVA)和双向ANOVA,并在适当时进行重复测量)。
可以执行Grubbs测试来识别异常值(Leggio 等人,2019,补充信息)。
延迟吃1个第一颗粒(在SE conds,d AY 小号17-18)经由双向重复测量(RM)方差分析典型地分析,以揭示主效应和相互作用(例如,基因型× 然后通过合适的日)交-hoc 测试,用于成对比较,例如Newman-Keuls测试。天达到标准(以天为单位,d AY 小号20-39)在训练阶段期间可通过不成对(两个取样值)t-检验或单向ANOVA揭示主效应(被分析例如根据,基因型或治疗)实验组的数量(两个以上)。最后,通常还会通过双向RM ANOVA分析最终阶段(第40天或更早)的正确选择的百分比(作为工作记忆的指标),以揭示主要影响和相互作用(例如,基因型× 延迟),然后进行适当的事后测试。
图3中的图表显示了进食延迟(A),达到标准的天数(B),以及在任务期间野生型(WT)和敲除小鼠同窝仔(此处为Dys +/- 小鼠)进行的正确选择(%,C)(改编自Leggio et al。,2019)。两组了解到类似奔驰进入迷宫来获取报酬,由延迟的显著降低所表明在第二天吃(d 着y 18)暴露于比第一天的奖励(d 着y 17) 。此外,两组都需要相同的天数才能连续3天达到正确选择的80%的标准。然而,尽管两组都表现出延迟依赖性行为(随着延迟时间的延长,错误逐渐增加),但在运行内延迟时间为4和30 s时,Dys +/- 小鼠比WT小鼠表现出明显的工作记忆缺陷。
 


D:\ Reformatting \ 2020-4-7 \ 1902963--1436 G.Leggio 828946 \ Figs jpg \ Fig。 3.jpg


图3. WT(n = 12)和Dys +/- (n = 10)小鼠在离散的成对配对可变延迟T迷宫任务中进行了测试。一。检索奖励的延迟(基因型F (1,20 )= 4.982,P = 0.0372; 天F (1,20 )= 68.04,P <0.0001,基因型× 天F (1,20 )= 5.37,P = 0.031; 事后:** P <0.01,相对于。第17天)。乙。达到标准所需的天数(P = 0.88)。Ç 。正确选择的百分比,具有不同的随机出现的运行中延迟(4、30、60 和240 s)和审判间隔20 s(基因型F (1,20 )= 5.95,P = 0.0241; 延迟F ( 3,60)= 14.93,P <0.0001,基因型× 延迟˚F (3,60)= 0.65,P = 0.58; 事后:* P <0.05,与。每个WT时间点)。这些数值是平均值±SEM (一个从Leggio dapted 等人,2019 )。


 


图4中的图形显示了通过聚类不同实验而获得的C57B16 / J WT小鼠(n = 46)的数据分布。延迟吃(A),天数的标准(B),和正确的选择(%,C),该C57BL6 / J野生型任务期间执行(一个从帕帕莱奥dapted 等人,2008 和2014; Leggio 等。,2019)。
 


D:\ Reformatting \ 2020-4-7 \ 1902963--1436 G.Leggio 828946 \ Figs jpg \ Fig 4.jpg


图4. 通过对不同实验进行聚类获得的C57B16 / J WT小鼠(n = 46)的数据分布。一。检索奖励的延迟(配对t 检验; *** P <0.001)。乙。d AYS需要达到的标准。Ç 。P 的不同随机赠送帧内运行延迟(4,30,60,和240秒)和20秒的审间延迟(正确选择ercentage 延迟˚F (3,180)= 22.49,P <0.0001)。值是平均值± SEM 。(一个从帕帕莱奥dapted 。等人,2008 和2014; Leggio 。等人,2019 )。


 


笔记


 


在整个实验过程中,动物都禁食以维持其90%的随意体重。维持90%的小鼠初始体重至关重要。不保持这种体重状况的小鼠趋向于减慢速度或不进入迷宫。
研究人员在此行为任务中应测试的理想小鼠数是10-12。在同一实验中测试更多的动物可能具有挑战性,因为日常过程的时间较长,主要是在测试的最后阶段(每只小鼠45-60分钟)。
在实验过程中,研究人员应避免将鼠标放在笼子的网格上。鼠标从家用笼中拿起并将其直接放置到迷宫的起始位置时,压力较小。
 


致谢


 


我们感谢M. Morini,D。Cantatore,R。Navone,G。Pruzzo,A。Parodi,A。Monteforte和C. Chiabrera的技术支持。这项工作得到了意大利技术大学,卡塔尼亚大学,脑与行为研究基金会(2015 NARSAD 23234)和西班牙圣保罗(2015-0321)的资助。所描述的方法先前已在Leggio 等人中使用。(2019)。


 


利益争夺


 


作者宣称他们没有竞争利益。


 


伦理


 


根据使用相关方法进行的研究选择动物样本大小,该样本大小与本领域中通常使用的大小相似。没有使用随机化将动物分配给实验组,研究者对动物的基因型视而不见。所有实验均根据欧盟指令2010/63 / EU以及卡塔尼亚大学和意大利理工学院(IIT)的机构动物护理和使用委员会进行。


 


参考资料


 


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引用:Leggio, G., Torrisi, S. A. and Papaleo, F. (2020). The Discrete Paired-trial Variable-delay T-maze Task to Assess Working Memory in Mice. Bio-protocol 10(13): e3664. DOI: 10.21769/BioProtoc.3664.
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