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Aug 2019

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Nestlet Shredding and Nest Building Tests to Assess Features of Psychiatric Disorders in Mice
巢破坏和筑巢测试评估小鼠精神障碍的特征   

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Abstract

Mimicking the various facets of human psychiatric and neurodevelopmental disorders in animal models is a challenging task. Nevertheless, mice have emerged as a widely used model system to study pathophysiology and treatment strategies for these diseases. However, the corresponding behavioral tests are often elaborate and require extensive experience in behavioral testing. Here, we present protocols for two simple assays, nest building and nestlet shredding, that can serve as a starting point for the behavioral phenotyping of mouse models with (potential) features of psychiatric disorders. Both tests have been reported previously and we extend prior descriptions by including adaptations and refinements derived from our practical experience, like the use of the home cage instead of a fresh cage for nestlet shredding. Summarized, we provide ready-to-use protocols for two behavioral assays that allow the generation of robust data with minimal time and cost expenditure and enable an initial assessment of features of psychiatric or neurodevelopmental disorders in mouse models of these diseases.

Keywords: Behavior (行为), Autism (孤独症), Neurodevelopmental disorder (神经发育障碍), Repetitive behavior (重复行为), Plasmalogen (缩醛磷脂), Stereotypy (刻板症), Stress (压力), Neurological disease (神经系统疾病), Mouse model (小鼠模型)

Background

Due to the unmatched complexity of the human brain, it is difficult to model features of psychiatric and neurodevelopmental diseases using animal models. However, in spite of some limitations, in mice an impressive battery of tests targeting numerous individual aspects of these disorders has been developed in recent years (Seong et al., 2002; Ricceri et al., 2007; Sukoff Rizzo and Crawley, 2017). Many of these assays are now widely accepted as valid tools to study disease mechanisms or therapeutic approaches. Here, we demonstrate two simple tests, nestlet shredding and nest building, widely used to study potential features of psychiatric and, particularly, neurodevelopmental disorders that we have – among others – used recently to characterize the behavioral phenotype of mice with a genetically caused deficiency to synthesize ether lipids (Dorninger et al., 2019a) (cf. Figure 1). The latter are compounds performing a wide range of biological activities, ranging from structural maintenance of cellular membranes to signal transduction (Dorninger et al., 2017a; Dorninger et al., 2020), and are particularly important for correct functioning of the brain (Berger et al., 2016). Accordingly, ether lipid-deficient mice present with complex behavioral abnormalities including motor disturbances, hyperactivity and memory deficits as well as restricted social interaction and interest in novel objects (Dorninger et al., 2017b; Dorninger et al., 2019a; Dorninger et al., 2019b).

Both of the presented assays have several obvious advantages: They are low-cost experiments, by which a significant amount of data can be obtained in a relatively short time frame. Furthermore, they can be reliably performed by investigators with only modest experience in behavioral analysis. Both tests are hardly dependent on physical abilities of the animals, thus allowing the use of genetically modified mouse models with marked phenotypes. For example, we used mice with considerable developmental defects and visual impairments (the ether lipid-deficient Gnpat knockout (KO) mouse model) (Rodemer et al., 2003), which precluded the application of many behavioral tests. However, as demonstrated by their performance in the nest building assay (Dorninger et al., 2019a), the animals are perfectly capable to utilize nestlets. Certainly, the simplicity of the assays also comes with some caveats: Both tests provide only an initial estimation on potential features of neurodevelopmental disorders and should not be used isolated. Also, they do not yield numeric data that can be directly related to the extent of human disease. Instead, they need to be complemented by additional behavioral tests assessing similar features (Silverman et al., 2010) and, optimally, embedded in a battery of assays allowing a comprehensive description of the behavioral phenotype. Furthermore, results need to be interpreted with care: Excessive nestlet shredding is generally seen as an indicator of repetitive behavior, a symptom also seen in human autism spectrum disorders or obsessive-compulsive disorder (Angoa-Perez et al., 2013). On the contrary, reduced engagement in the task may be viewed as restricted interest in novel objects, another feature commonly associated with autism (Bernard et al., 2015; Dorninger et al., 2019a). We feel that both interpretations are valid but need to be considered in the context of other aspects of behavior of a certain mouse model. Nest building, in turn, can be indicative of general well-being, distress or pain (Jirkof, 2014). At the same time, impairments in this task could hint towards structural or functional deficits in the brain, as demonstrated by mouse models of brain injury or with genetic modifications (Lijam et al., 1997; Deacon et al., 2002; Sager et al., 2010).

Our procedures for both tests are based on the previous protocols (Deacon, 2006; Angoa-Perez et al., 2013; Neely et al., 2019) that we have slightly modified. Furthermore, we provide more detailed descriptions of the testing protocols, including novel recommendations on the testing environment and extended illustration of the procedures and results, and share our experiences in test execution and analysis.

Materials and Reagents

  1. Cotton fiber nestlets (Ancare; product no. NES3600 ; 1 per mouse and behavioral test)

  2. Labeled plastic bags for storage of used nestlets

  3. Animals

    There is no restriction concerning the mouse strain to be used for the current protocols. The presented data were generated using animals on an outbred C57BL/6 x CD1 background carrying a targeted inactivation of the Gnpat gene (Gnpattm1Just, MGI: 2670462 ; Rodemer et al., 2003) and their wild type littermates.

Equipment

  1. Common laboratory balance (minimum accuracy: 0.1 g)

  2. Clean mouse cages with fresh bedding (as conventionally used by your animal facility)

  3. Standard camera

  4. Timer

  5. Forceps

  6. Glass or plexiglass plate; dimensions depending on the size of the cage used for testing (the plate should be large enough to replace the cage lid)

Software

  1. Standard statistics software: Prism (GraphPad; version 6 or higher), Sigma Plot (Systat; version 11.0 or higher) or similar

  2. Optional: Video tracking software (e.g., VideoTrack, ViewPoint or similar)

Procedure

  1. Nestlet shredding test

    1. House mice individually for at least 7 days prior to the test. Make sure that housing conditions fulfill common standards like controlled lighting (in our case 12:12 h light-dark), temperature and humidity as well as low noise level, enriched cage environment and provision of chow and water ad libitum.

    2. 24 h prior to the test, remove any material for nesting and environmental enrichment from the cage of test mice.

    3. On the testing day, relocate testing animals to the test area. Make sure the animals are accustomed to their environment to avoid distraction, for example by unfamiliar smells. This can be achieved by performing the test in a separated space of the room, in which the mice are housed. Alternatively, habituate mice to the test area on the days prior to testing for a minimum of 1 h per day.

    4. Remove food and water from the test animal’s cage and replace the lid by a transparent object with a smooth surface, such as a (glass or plexiglass) plate, to prevent mice from climbing during the test time.

    5. Take a nestlet using a forceps, weigh it and place it in the test animal’s cage.

    6. Ensure documentation by taking a photo at the beginning of the trial. Alternatively, an automated video system (e.g., VideoTrack, ViewPoint or similar) may be used. Make sure to document identifying information of the tested animal (e.g., by picturing the cage label including the mouse ID prior to picturing the nestlet).

    7. Start the timer. During the observation period of 30 min, keep a distance of at least 1 m from the test area.

    8. After 30 min, stop the trial and gently remove the test animal from the cage. Make sure all nestlet material remains in the cage.

    9. Document the state after the end of the trial using the camera (Figure 1).

    10. Remove the nestlet and weigh it. If the nestlet is torn into several pieces, use the largest one. Do not include pieces that fall off during removal of the nestlet, but do not deliberately try to shake off loose material.

    11. Store the nestlet in a plastic bag for documentation. Do not forget to label the bag with the mouse ID.

    12. When testing animals sequentially, alternate between treatment groups to avoid bias in your analysis.



      Figure 1. Nestlet shredding in a cohort of wild type and ether lipid-deficient (“Gnpat KO”) mice. A. Nestlets after the 30 min-observation period are documented for all tested animals (n = 9 per genotype). Numbers inside the panels indicate the test ID (black: wild type; red: Gnpat KO). Note the markedly higher shredding activity of wild type compared with Gnpat KO mice. B. The difference in nestlet weight before and after the trial was calculated for each test animal and results are shown as individual data points. Grey bars indicate the mean value. Numbers next to the data points designate the mouse test ID, as defined in (A). Statistical testing was performed using Mann-Whitney U-test (**p < 0.01). The quantitative data shown here have also been presented in our previous publication ( Dorninger et al., 2019a).


  2. Nest building test

    1. House mice individually for at least 7 days prior to the test. Make sure that housing conditions fulfill common standards like controlled lighting (in our case 12:12 h light-dark), temperature and humidity as well as low noise level, enriched cage environment and provision of chow and water ad libitum. Testing can be performed in the animals’ standard housing room, which ensures maximal familiarization of mice with their environment.

    2. 24 h prior to the test, remove any nesting material from the cage of test mice.

    3. Avoid any setting that could provoke a bias in your test results (e.g., spatially separating different test groups).

    4. On the test day (preferably in the evening), supply every test mouse with a nestlet. Distribute nestlets using a clean forceps.

    5. Document the state at the beginning of the trial by taking a photo.

    6. After 20 h, document the status quo (“t20”). Take care not to disorganize the nest – thus, avoid any agitation of the animals. Therefore, to take a photo, wait till the mouse either sits steadily in its nest (make sure that the nest is clearly visible) or leaves its nest voluntarily. Do not dislocate mice forcibly from the nest. Make sure to document identifying information of the tested animal (e.g., by picturing the cage label including the mouse ID prior to picturing the nestlet).

    7. After 48 h, end the trial by moving test mice to a clean cage. Document the final nest and avoid disorganizing it.

Data analysis

  1. Nestlet shredding test

    1. For every test animal, calculate the difference between the nestlet weight before and after the trial (Figure 1).

    2. Do not exclude animals, which did not engage in nestlet shredding. In general, we recommend to make use of exclusion sparingly (if a statistical exclusion criterion is chosen, use > 5-7 x SD; Barbato et al., 2011). We usually take the position that uncommon behavior in a certain behavioral task is insufficient to exclude animals, as this may create a bias in your analysis. Thus, only exclude animals that are obviously impaired (e.g., hurt) or physically unable to perform the task. However, this should be done prior to performing the trial, in compliance with current ARRIVE guidelines (Percie du Sert et al., 2020).

    3. Compare your test groups using the statistics software of your choice. In the case of normally distributed values, parametric tests may be used (e.g., Student’s t-test for comparison of two groups, one-way ANOVA for comparison of more than two groups). However, with the mouse strain that we used, a considerable number of values equaled to 0, thus precluding the use of parametric tests. In that case, use non-parametric tests like the Mann-Whitney U-test (for comparison of two groups) or the Kruskal-Wallis test (for comparison of more than two groups).


  2. Nest building test

    1. For the assessment of nests, engage 2-3 independent investigators that were not involved in the testing procedure or in handling the mice.

    2. Do not exclude animals, which did not engage in nest building. If any other exclusion criteria are applied, make sure to follow the ARRIVE guidelines (Percie du Sert et al., 2020).

    3. Grade nests using scores ranging from 1 (very poor/no nest building) to 5 (optimal nest building) according to a previously published scoring scheme (Deacon, 2006) (Table 1 and Figure 2). Assessors must be blinded to the genotype or treatment group of the test animals.


      Table 1. Scoring scheme for assessment of nests, as described by Deacon, 2006. Note that grading steps of 0.5 can be assigned for more exact evaluation.



      Figure 2. Scoring in the nest building test. Scoring was performed according to a previously published assessment scheme (Table 1; Deacon, 2006) using grades ranging from 1 (very poor) to 5 (optimal). Examples of nests reaching scores of 2 (A), 3 (B), 4 (C) and 5 (D) are shown. For all of these nests, the vote of two independent evaluators was unanimous. Note that grade 1 would correspond to an untouched nestlet. However, no animal graded lower than a score of 2 in our test cohort.


    4. Use non-parametric tests for comparison of your test groups at each time point. Correction for multiple testing at the two time points should be considered (in particular, if analysis at t20 and at t48 do not target different research questions).

Notes

  1. Like for any behavioral assay, make sure you test all animals at approximately the same time of the day, preferably in the evening, which marks the beginning of the active phase of mice when housed at a conventional light-dark cycle.

  2. In previous protocols, nestlet shredding was described in clean cages with fresh bedding (Angoa- Perez et al., 2013). However, in the mouse strain we used, this led to increased interest of the animals in the unfamiliar environment and less engagement in nestlet shredding. To avoid this kind of distraction, we performed the assay in the animals’ home cage, which clearly increased shredding activity and generated more discriminative results (Figure 3).



    Figure 3. Different nestlet shredding activity in the home cage versus a fresh cage. The same cohort of mice (n = 8) was exposed to the nestlet shredding test in a cleaned cage filled with fresh bedding (“fresh cage”) and, one week later to exclude habituation to the task, in their home cage. Connected data points derive from the same test animal. Note that the same shredding score (0 g in the fresh cage and 0.1 g in the home cage) was reached by three different mice. Statistical analysis was performed using paired Student’s t test. *p < 0.05


  3. Additional information can be gathered by repeating the nestlet shredding assay on consecutive days. However, for our purposes, we decided against repeated measurements to avoid habituation to the task.

  4. We tested several types of nesting material for use in the nest building test. In our hands, nestlets proved to produce most reproducible results and led to a more discriminative performance compared with paper towels, customary kitchen roll (cut in stripes or uncut) or cotton wool (Figure 4). Please note that other authors suggested to conduct four consecutive trials with different types of material (Neely et al., 2019), which may be considered as an alternative option.



    Figure 4. Comparison of different material types for use in the nest building test. Test animals were supplied with a sheet of customary kitchen roll (either uncut, A, or cut to stripes, B), a paper towel (cut to stripes, C), cotton wool (D) or a nestlet (E) and the nests produced after a trial period of 24 h are shown. For the present protocol, the nestlet was selected as the most suitable material, as it produced the most discriminative results.


  5. The time points, at which nests are analyzed in the nest building test, can be varied at the experimenter’s discretion. However, make sure to cover at least one overnight period, as this constitutes the animals’ active phase, i.e., we recommend a minimum of 12 h until the first analysis.

  6. Mice of both sexes can be used for the presented tests. To minimize variability, male and female mice should be analyzed separately. However, keep in mind that in this case statistical correction for multiple comparisons may have to be performed.

  7. Similarly, there are only limited constraints concerning the age of the test animals. Basically, adult animals of any age can be used. Yet, we generally recommend to keep the age range as narrow as possible for low variability. Also, the phenotype of genetically modified models may change with age, thus affecting performance in behavioral assays.

  8. Both tests can be performed with the same cohort of animals. However, in order to avoid habituation to the nestlets, a resting interval of 7 days should be maintained between the tests.

  9. In our experiments, we used mice of a mixed background strain (outbred C57BL/6 x CD1). These animals reliably showed nesting behavior. However, nesting has been reported to be highly strain-sensitive (Deacon, 2006 and 2012), thus baseline data should be obtained in a test cohort of your background strain. Average nestlet shredding activity in our strain was similar as described previously (Angoa-Perez et al., 2013). Yet, we experienced considerable variability (Dorninger et al., 2019a), thus emphasizing the need for statistical power calculation using baseline data before performing the assay in your test cohort.

  10. In case you are working with a mouse strain/genotype/treatment condition that does not produce good nests, the percentage of nestlet material used for nest building can be taken as an alternative readout parameter. For this, weigh the nestlet prior to supplying it to the mice (step 4). After the trial, weigh the part of the nestlet that was not utilized for nest building and calculate the fraction of nestlet material that has been converted to the nest.

Acknowledgments

This work was supported by the Austrian Science Fund (FWF, P24843-B24, P31082-B21 and I2738-B26) and RhizoKids International. Data derived from the use of the protocols described here have been published recently ( Dorninger et al., 2019a).

Competing interests

The authors declare no competing interests.

Ethics

All described protocols and experiments were approved by the Institutional Animal Care and Use Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research (BMWF-5.011/0003-II/10b/2009 and BMBWF-66.009/0174-V/3b/2019).

References

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简介

[摘要]在动物模型中模仿人类精神疾病和神经发育疾病的各个方面是一项艰巨的任务。然而,小鼠已经成为一种广泛使用的模型系统来研究这些疾病的病理生理和治疗策略。但是,相应的行为测试通常很复杂,并且需要在行为测试方面有丰富的经验。在这里,我们为两种简单的检测方法(巢构建和巢切碎)提供方案,可以作为具有(潜在)精神疾病特征的小鼠模型的行为表型的起点。先前已经报道了这两种测试,并且我们通过包括从我们的实际经验中得出的修改和改进来扩展先前的描述,例如使用家用笼子代替新鲜的笼子来切碎巢。综上所述,我们为两种行为分析提供了现成的协议,这些协议可以以最少的时间和成本支出生成可靠的数据,并可以初步评估这些疾病的小鼠模型中的精神病或神经发育障碍的特征。


[背景]由于人脑无与伦比的复杂性,很难使用动物模型来模拟精神病和神经发育疾病的特征。然而,尽管有一些局限性,但近年来在小鼠中针对这些疾病的许多个体方面进行了一系列令人印象深刻的测试(Seong等人,2002; Ricceri等人,2007; Sukoff Rizzo和Crawley,2017) 。这些分析方法中的许多方法现在已被广泛认为是研究疾病机制或治疗方法的有效工具。在这里,我们演示了两个简单的测试,即切碎巢和筑巢,广泛用于研究精神病的潜在特征,尤其是神经发育障碍,我们最近(其中包括)用于表征具有遗传缺陷的小鼠的行为表型。合成醚脂质(Dorninger等,2019a)(参见图1)。后者是具有广泛生物学活性的化合物,范围从细胞膜的结构维护到信号转导(Dorninger等,2017a; Dorninger等,2020),对于大脑的正确功能特别重要(Berger等人,2016)。因此,缺乏醚脂质的小鼠表现出复杂的行为异常,包括运动障碍,活动过度和记忆力低下,以及社交互动受到限制以及对新对象的兴趣(Dorninger等人,2017b; Dorninger等人,2019a; Dorninger等人。,2019b)。

所提出的两种检测方法都有几个明显的优点:它们是低成本的实验,通过这些实验可以在相对较短的时间内获得大量数据。此外,只有行为分析方面的经验很少的调查人员才能可靠地执行这些操作。两种测试都几乎不依赖于动物的身体能力,因此可以使用具有明显表型的转基因小鼠模型。例如,我们使用了具有相当大的发育缺陷和视觉障碍的小鼠(醚脂质不足的Gnpat基因敲除(KO)小鼠模型)(Rodemer等,2003),这排除了许多行为测试的应用。然而,正如它们在筑巢试验中的表现所证明的那样(Dorninger等,2019a),这些动物完全有能力利用巢穴。当然,这些测定方法的简单性还带有一些警告:两种测试方法仅提供了神经发育障碍潜在特征的初步估计,因此不应孤立使用。而且,它们不会产生与人类疾病程度直接相关的数值数据。取而代之的是,它们需要通过评估相似特征的其他行为测试来补充(Silverman等人,2010),并且最好嵌入到一系列能够对行为表型进行全面描述的测定中。此外,必须谨慎地解释结果:过度的巢状切碎通常被视为重复行为的指标,也是人类自闭症谱系障碍或强迫症的症状(Angoa-Perez等,2013)。相反,减少从事这项工作可被视为对新颖对象的有限兴趣,这是与自闭症通常相关的另一个特征(Bernard等,2015; Dorninger等,2019a)。我们认为这两种解释都是有效的,但需要在某些鼠标模型的行为的其他方面进行考虑。反过来,筑巢可以预示一般的幸福感,痛苦或痛苦(Jirkof,2014)。同时,这项工作的障碍可能暗示大脑的结构或功能缺陷,如小鼠脑损伤模型或遗传修饰所证明的那样(Lijam等,1997; Deacon等,2002; Sager等。 。,2010)。

我们对两个测试程序是基地d上的先前协议(执事,2006; Angoa -佩雷斯等人,2013;尼利等人,2019),我们稍微修改。此外,我们提供了有关测试协议的更详细描述,包括对测试环境的新颖建议以及对过程和结果的扩展说明,并分享了我们在测试执行和分析方面的经验。

关键字:行为, 孤独症, 神经发育障碍, 重复行为, 缩醛磷脂, 刻板症, 压力, 神经系统疾病, 小鼠模型


材料和试剂
 
1.棉纤维巢(Ancare;产品编号NES3600;每只小鼠1只和行为测试)      
2.贴有标签的塑料袋,用于存放用过的巢      
3.动物      
对于当前协议要使用的鼠标株系没有限制。所呈现的数据是使用带有目标失活Gnpat基因的远交C57BL / 6 x CD1背景上的动物生成的(Gnpat tm1Just ,MGI:2670462; Rodemer et al。,2003)及其野生型同窝动物。
 
 
设备
 
普通实验室天平(最低精度:0.1 g )
用新鲜的被褥清洁鼠标笼(通常由动物设施使用)
标准相机
计时器
钳子
玻璃或有机玻璃板;尺寸取决于用于测试的笼子的尺寸(板应足够大以更换笼子的盖子)
 
软件
 
标准统计软件:Prism(GraphPad;版本6或更高版本),Sigma Plot(Systat;版本11.0或更高版本)或类似版本
可选:视频跟踪软件(例如VideoTrack,ViewPoint或类似软件)
 
程序
 
Nestlet粉碎测试
在测试之前,至少要单独饲养小鼠至少7天。确保住房条件符合通用标准,例如受控照明(在我们的情况下为12:12 h暗),温度和湿度以及低噪音水平,丰富的笼舍环境和随意提供的食物和水。
测试前24小时,从测试小鼠的笼子中取出任何用于筑巢和丰富环境的材料。
在测试当天,将测试动物转移到测试区域。确保动物习惯于自己的环境,以避免分散注意力,例如因不熟悉的气味而分散注意力。这可以通过在容纳老鼠的房间的单独空间中执行测试来实现。或者,在每天测试至少1小时之前,将小鼠习惯于测试区域。
从测试动物的笼子中取出食物和水,并用光滑表面的透明物体(例如玻璃板或有机玻璃板)盖上盖子,以防止小鼠在测试时间内攀爬。
用镊子取一窝,称重,然后放在测试动物的笼子里。
在试验开始时通过拍照确保文档记录。备选地,可以使用自动视频系统(例如,VideoTrack,ViewPoint或类似物)。确保记录被测动物的识别信息(例如,在描绘巢之前,先描绘笼子标签,包括鼠标ID)。
启动计时器。在30分钟的观察期内,离测试区域至少1 m的距离。
30分钟后,停止试验并从笼子中轻轻移出试验动物。确保所有巢状材料保留在笼中。
使用摄像机记录试验结束后的状态(图1)。
取下巢穴并称重。如果将嵌套件撕成几块,请使用最大的一个。切勿包括在去除巢穴期间掉落的碎片,但不要故意甩掉松散的物质。
将嵌套料存放在塑料袋中以备说明。不要忘了用鼠标ID在包装袋上贴标签。
依次测试动物时,请在治疗组之间轮流使用,以避免分析时出现偏差。
 
图1.在一组野生型和醚脂缺乏型(“ Gnpat KO”)小鼠中切碎小巢。A.观察30分钟后的所有实验动物的巢穴都有记录(每基因型n = 9)。面板内部的数字表示测试ID(黑色:野生型;红色:Gnpat KO)。请注意,与Gnpat KO小鼠相比,野生型的切碎活性明显更高。B.计算每只试验动物试验前后巢重的差异,结果显示为单独的数据点。灰色条表示平均值。数据点旁边的数字表示(A)中定义的鼠标测试ID。使用Mann-Whitney U检验进行统计检验(** p <0.01)。此处显示的定量数据也已在我们之前的出版物中提出(Dorninger等,2019a)。  
 
筑巢测试
在测试之前,至少要单独饲养小鼠至少7天。确保住房条件符合通用标准,例如受控照明(在我们的情况下为12:12 h暗),温度和湿度以及低噪音水平,丰富的笼舍环境和随意提供的食物和水。可以在动物的标准饲养室内进行测试,以确保小鼠对其环境的最大程度的了解。
测试前24小时,从测试小鼠的笼子中取出任何嵌套材料。
避免任何可能引起测试结果偏差的设置(例如,在空间上分隔不同的测试组)。
在测试当天(最好是晚上),为每只测试鼠标提供一个Nestlet。使用干净的镊子散布巢。
通过拍照记录试验开始时的状态。
20小时后,记录现状(“ t20”)。注意不要弄乱巢穴-因此,避免对动物进行任何搅动。因此,要拍照,请等到鼠标稳固地坐在其巢中(确保该巢清晰可见)或自愿离开其巢为止。不要强行将小鼠从巢中移开。确保记录被测动物的识别信息(例如,在描绘巢之前,先描绘笼标签,包括鼠标ID)。
48小时后,将试验小鼠移至干净的笼子中以结束试验。记录最终的嵌套,并避免使其混乱。
 
数据分析
 
Nestlet粉碎测试
对于每只测试动物,计算试验前后的巢重之间的差异(图1)。
不要排除没有进行巢状切碎的动物。通常,我们建议您尽量少使用排除功能(如果选择了统计排除标准,请使用> 5-7 x SD; Barbato等人,2011)。我们通常会认为某些行为任务中的罕见行为不足以排除动物,因为这可能会在您的分析中造成偏差。因此,仅排除明显受损(例如受伤)或身体上无法执行任务的动物。但是,这应该在进行试验之前按照当前的ARRIVE指南进行(Percie du Sert等,2020)。
使用您选择的统计软件比较您的测试组。在正态分布值的情况下,可以使用参数检验(例如,用于比较两组的Student t检验,用于比较两组以上的单向方差分析)。但是,对于我们使用的鼠标应变,相当数量的值等于0,因此排除了参数测试的使用。在这种情况下,请使用非参数检验,例如Mann-Whitney U检验(用于两组比较)或Kruskal-Wallis检验(用于两组以上进行比较)。
 
筑巢测试
为了评估巢,请聘用2-3名独立的调查员,他们不参与测试程序或处理老鼠。
不要排除没有参与筑巢活动的动物。如果应用了其他任何排除标准,请确保遵循ARRIVE准则(Percie du Sert等,2020)。
根据先前发布的评分方案(Deacon,2006年),使用从1(非常差/没有筑巢)到5(最佳筑巢)的分数对巢进行评分(表1和图2)。评估者必须不知道测试动物的基因型或治疗组。
 
表1.如Deacon,2006年所述,用于巢穴评估的评分方案。请注意,可以指定0.5级的评分以进行更精确的评估。 
 
 
图2.筑巢测试中的评分。根据先前公布的评估方案(表1; Deacon,2006)进行评分,评分范围从1(非常差)到5(最佳)。显示了达到分数2(A),3(B),4(C)和5(D)的巢的示例。对于所有这些巢穴,两位独立评估员的投票都是一致的。请注意,等级1对应于不变的嵌套。但是,在我们的测试队列中,没有动物的评分低于2分。
 
使用非参数测试在每个时间点比较测试组。应该考虑在两个时间点进行多次测试的校正(特别是,如果在t20和t48的分析没有针对不同的研究问题)。
 
笔记
 
像进行任何行为分析一样,请确保您在一天的大约同一时间(最好在晚上)测试所有动物,这标志着按常规的明暗循环饲养时小鼠活动期的开始。
在以前的协议中,描述了在干净的笼子中用新鲜的被褥切碎鸟巢(Angoa-Perez等人,2013)。但是,在我们使用的小鼠品系中,这导致了动物在陌生环境中的兴趣增加,并减少了巢切碎的参与。为避免这种干扰,我们在动物的家笼中进行了测定,该测定明显增加了切碎活性并产生了更多的判别结果(图3)。
 
 
图3.家用笼子和新鲜笼子中不同的嵌套切碎活动。同一组小鼠(n = 8)在装满新鲜被褥的干净笼子(“新鲜笼子”)中进行巢状切碎测试,一周后将其习惯化以排除在其家笼中。连接的数据点源自同一只测试动物。请注意,三只不同的小鼠达到了相同的切碎分数(新鲜笼中为0 g,家庭笼中为0.1 g)。使用成对的学生t检验进行统计分析。* p <0.05
 
通过在连续的几天中重复进行巢状切碎测定,可以收集更多信息。但是,出于我们的目的,我们决定不重复测量,以避免习惯任务。
我们测试了几种用于筑巢测试的筑巢材料。与纸巾,常规厨房用纸卷(切成条状或未切开的)或棉绒相比,在我们手中,嵌套被证明可产生最可重复的结果,并具有更具区分性的性能(图4)。请注意,其他作者建议使用不同类型的材料进行四次连续试验(Neely et al。,2019),可以将其视为替代方案。
 
 
图4.用于筑巢测试的不同材料类型的比较。给测试动物提供一张习惯性的厨房用纸(未切开,A或切成条纹,B),纸巾(切成条纹,C),纯棉(D)或巢(E)和巢显示了24小时的试用期后生产的产品。对于本协议,选择Nestlet作为最合适的材料,因为它产生了最有区别的结果。
 
在筑巢测试中分析巢穴的时间点可以由实验人员自行决定。但是,请确保至少覆盖一个晚上,因为这构成了动物的活跃期,即,我们建议在首次分析之前至少保留12小时。
双方的小鼠都可以用于提出的测试。为了最大程度地减少变异性,应分别分析雄性和雌性小鼠。但是,请记住,在这种情况下,可能必须执行多次比较的统计校正。
类似地,关于测试动物的年龄只有有限的限制。基本上,可以使用任何年龄的成年动物。但是,我们通常建议将年龄范围保持尽可能窄,以降低变异性。同样,基因改造模型的表型可能会随着年龄的增长而变化,从而影响行为分析的性能。
可以对同一组动物进行两种测试。但是,为了避免巢状的习惯,试验之间应保持7天的休息时间。
在我们的实验中,我们使用了混合背景菌株的小鼠(杂种C57BL / 6 x CD1)。这些动物可靠地表现出筑巢行为。但是,据报道嵌套对应变非常敏感(Deacon,2006年和2012年),因此应该在测试背景菌株的队列中获得基线数据。我们菌株中的平均巢状切碎活性与以前描述的相似(Angoa-Perez等,2013)。但是,我们经历了相当大的可变性(Dorninger等人,2019a),因此强调了在您的测试队列中执行测定之前需要使用基准数据进行统计功效计算的需求。
如果您使用的小鼠品系/基因型/治疗条件不能产生良好的巢,则可将用于构建巢的巢材料的百分比用作替代的读出参数。为此,在将其供应给小鼠之前,先将其称重(步骤4)。试用后,称量尚未用于筑巢的Nestlet的部分,并计算已转换为Nest的Nestlet材料的比例。
 
 
致谢
 
这项工作得到了奥地利科学基金(FWF,P24843-B24,P31082-B21和I2738-B26)和RhizoKids International的支持。最近已发布了使用此处描述的协议得出的数据(Dorninger等,2019a)。
 
利益争夺
 
作者宣称没有利益冲突。
 
伦理
 
所描述的所有方案和实验均得到维也纳医科大学的机构动物护理和使用委员会以及奥地利联邦科学与研究部(BMWF-5.011 / 0003-II / 10b / 2009和BMBWF-66.009 / 0174-V)的批准/ 3b / 2019)。
 
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引用:Dorninger, F., Zeitler, G. and Berger, J. (2020). Nestlet Shredding and Nest Building Tests to Assess Features of Psychiatric Disorders in Mice. Bio-protocol 10(24): e3863. DOI: 10.21769/BioProtoc.3863.
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