Jun 2018



A Single Test to Study Social Behavior and Repetitive Self-grooming in Mice

引用 收藏 提问与回复 分享您的反馈 Cited by


The ability to recognize and interact with members of the same species is essential for social communication. Investigating the neural substrates of social interest and recognition may offer insights into the behavioral differences present in disorders affecting social behavior. Assays used to study social interest in rodents include the 3-chamber test, a partition test, and a social interaction test. Here, we present a single protocol that can be used to quantify the level of social interest displayed by mice, the ability to distinguish between different individual mice (social recognition), and the level of repetitive self-grooming displayed. In the first part of the protocol, a social habituation/dishabituation test, the time spent by a test mouse sniffing a stimulus mouse is quantified over 9 trials. In the first 8 interactions, the same stimulus mouse is used repeatedly; on the ninth trial, a novel stimulus mouse is presented. Intact social recognition is indicated by a progressive decrease in the investigation time over trials 1-8, and an increase in trial 9. The interval between each social trial is used to quantify self-grooming, a stereotyped repetitive behavior in mice. We also present a method for randomized, blinded analysis of these behaviors to increase rigor and reproducibility of results. Therefore, this single behavioral test enables ready assessment of phenotypes of both social and repetitive behaviors in an integrated manner in the same animals. This feature can be advantageous in understanding interactions between these behaviors and phenotypes in mouse models with genetic variants associated with autism and other neurodevelopmental or neuropsychiatric disorders, which are often characterized by these behavioral differences.

Keywords: Behavior (行为), Habituation (习惯化), Dishabituation (去习惯化), Grooming (理毛), Blinded analysis (盲态分析), Social interest (社交兴趣), Social recognition (社会识别)


Verbal and nonverbal communications enhance an organism’s ability to establish and maintain a social hierarchy, and thus survive and reproduce. These forms of communication rely on the motivation to interact with fellow conspecifics and the ability to recognize and distinguish between different individuals. In humans, disorders that are commonly characterized by altered social behavior include depression, anxiety, and autism (Saunders and Roy, 1999; White et al., 2009). Although there is a general understanding of symptoms, the etiologies of these disorders, particularly the underlying mechanisms of social impairments and differences, remain unclear (Goldstein and Rosselli, 2003).

Mice offer advantageous models for studying the neurobiology of behavior through similarities to certain aspects of human behavior and physiology (Austin et al., 2004; Cryan and Holmes, 2005). Although a mouse model will not perfectly replicate human disease, overall symptoms can apply when investigating theories regarding genetic or environmental modulation (Crawley, 2007). For example, much of the current preclinical autism research revolves around the diagnostic criteria of abnormal social interaction, impaired social communication and interaction, and repetitive behaviors (Silverman et al., 2010).

A social habituation/dishabituation test enables measurement of two components of rodent social behavior: 1) the interest in a familiar social stimulus shown repeatedly over a short period of time; and 2) the ability to recognize a new social stimulus (Tejada and Rissman, 2012; Ujjainwala et al., 2018). In this paradigm, the mouse used to provide a social stimulus is placed under a wire cup to confine it to a fixed location and to physically separate it from the mouse whose behavior is being examined (“test mouse”). Interest is measured by the amount of time the test mouse spends exploring the stimulus mouse under the wire cup, typically quantified through sniffing. This paradigm can be used to investigate biological and environmental influences on social interest and/or social recognition. For example, our group recently reported that mice genetically deficient for the diazepam binding inhibitor (DBI) protein show a significant reduction in social interest compared to wild-type littermates, with no deficit in social recognition (Ujjainwala et al., 2018). Furthermore, rodents often show sex differences in social interest levels (Tejada and Rissman, 2012; Karlsson et al., 2015), and sex steroid hormones, such as testosterone and estradiol, affect social exploratory behavior. Variations of this protocol have also been used to examine the loss of the oxytocin gene as well as lesions to the CA2 region of the hippocampus and their effect on social recognition (Ferguson et al., 2000; Stevenson and Caldwell, 2014). In addition, this test could be applied to further evaluate the impacts of other environmental and behavioral factors, such as social isolation or gestational exposure to bisphenol A (Wolstenholme et al., 2012; Lander et al., 2017).

Restricted repetitive behaviors are most often associated with autism in humans, but these behaviors are also present in other disorders such as Rett, Fragile X, and Prader-Willi syndromes (Crawley, 2007; McFarlane et al., 2008; Lewis and Kim, 2009). In mice, this phenotype can present in numerous forms, including repetitive grooming, circling, jumping, or other stereotypical movements (Crawley, 2007). Given the overlap between impaired social interaction and repetitive behaviors in these and other neuropsychiatric and neurodevelopmental disorders, a single protocol that enables ready and integrated assessment of multiple phenotypes is advantageous.

Another paradigm commonly used to study social interest and recognition in mice is the 3-chamber test. This test requires a specialized apparatus containing multiple chambers separated by removable doors. This configuration is of higher novelty to a mouse than an environment that mirrors the home cage, potentially inducing higher levels of general exploratory behavior rather than social interaction specifically (Yang et al., 2011). By contrast, the test described in this protocol can be performed in a standard mouse cage used for daily housing in many laboratories. The other materials required can be obtained readily at a low cost.

Blinded analyses significantly enhance experimental rigor, especially regarding manual scoring of behavior, where pre-conceptions may lead to biased evaluation. For any given study, requiring separate researchers to either conduct experiments or perform analyses is not always feasible and can lead to differences in interpretation. Here, we provide a method to randomly code, name, and sort files using Excel. This method allows for one person to conduct the behavioral test and then score the videos in a blinded fashion. This process could be used to anonymize other file types for blinded analysis as well.

Materials and Reagents

  1. Chrome Round Pencil Holder (Spectrum Diversified, catalog number: 31570, diameter of 10.2 cm, distance between bars of 0.64 cm, initial height of 10.8 cm)
  2. 400 ml plastic beaker
  3. Post-pubertal male or female test mice of any strain
  4. 2 stimulus mice (e.g., ovariectomized adult female mice)
    Note: For this procedure, at least two stimulus mice are needed. Ensure both stimulus mice are of the same sex and age, and that both have had no prior contact with any of the test mice (i.e., not littermates). Stimulus mice can be either males or females as they are physically separated during the test with the cup. Commonly, ovariectomized females are used to provide neutral stimuli to quantify social interest without provoking aggressive or sexual responses. Consult with local animal care committee guidelines regarding ovariectomy surgeries if needed.
  5. Ethyl Alcohol 70%
  6. Deionized (DI) water


  1. MiliporeSigma Synergy Ultrapure Water Purification System (MiliporeSigma, model: SYNSOHFUS)
  2. Windows PC computer
  3. Video Camera (Logitech Webcam C930e, 90° field of view, 1920 x 1080 pixels)
    Note: Any camera with similar specifications that can visualize that entire cage can be used for this protocol.
  4. Testing chamber (bottom and filter top of standard mouse cage can be used with the dimensions of 29.3 cm x 17.1 cm x 12.7 cm for the base, and a total height of 20.3 cm with the lid placed on top)
  5. Corn Cob Bedding (use the same type of bedding as in the home cage)


  1. iSpy (freeware open-source video surveillance platform available at www.ispyconnect.com)
  2. Microsoft Excel
  3. Statistics software, such as SAS


  1. Setup of clean testing chamber and wire cup
    1. Line a clean mouse cage bottom with new bedding to a depth of 1-2 cm. The cage used should be equivalent to the type typically used for housing. Food and water should not be present in the testing chamber.
    2. Clean a wire cup with 70% EtOH, followed by DI water, ensuring it is completely cleaned and dry before placement in the testing chamber. The wire cup should have a diameter and height large enough to accommodate one mouse.
      Note: If the wire cup is not tall enough to prevent the mouse from climbing and perching on top, extend the height of the wire cup by affixing the bottom of a 400 ml plastic beaker to the underside of the wired cup with glue or tape, as shown in the Figure 1. An overall cup height of > 14 cm should prevent most mice from perching on the top of the cup.

      Figure 1. Example of extended stimulus cup. To prevent the mouse from climbing on top, the height of the cup can be extended by attaching the bottom of a 400 ml plastic beaker. This cup is extended by 3.75 cm to a total height of 14.6 cm. A height of at least 14 cm is typically sufficient to prevent a mouse from perching on top.

    3. The testing area should be isolated from other laboratory activity. If a separate room is not available, isolate the testing area by hanging opaque sheets around it and keep the room as quiet as possible to minimize disruption.
    4. Within the testing area, the lights should be dim, and there should not be any bright light directly above, as light level can influence exploratory behaviors in mice (Morato and Castrechini, 1989). Use a light level (approximately 3-6 lux) sufficient to visualize the entire cage and the test mouse through the video camera. This illumination can be achieved through indirect light.

  2. Setup of camera and computer for video recording
    1. Position camera to ensure it captures the entire area of the testing chamber. The camera should be placed perpendicular to the long end of the cage. The camera should be close enough for detailed quantification, but far enough away that the entire cage can be visualized (Figure 2).
    2. Connect the camera to the video software iSpy.
    3. Edit the camera settings to set the recording time to 610 s (10 min plus 10 s), the max frame rate to 10 FPS, and the buffer time to 2 s. Each trial is 10 min long; the additional 10 s are used to place the stimulus mouse within the cup.

      Figure 2. Testing area setup including testing chamber, camera, stimulus cup, and opaque sheets for light control. In this setup, the lights directly over the cage are turned off, so the light in the testing area comes from the other side of the opaque sheets. Laboratory tape is placed on the bench to indicate consistent placement of the cage.

  3. Acclimatization of mice to testing room and chamber
    1. Bring stimulus and test mice to the testing room in their respective home cages. During transport, cages should be individually covered by opaque cloths or dark plastic bags. Keep mice in home cages until ready for individual acclimatization to the testing chamber. Cages holding stimulus mice and test mice should be placed at least 1.5 m away from each other to prevent any interaction prior to the test.
    2. Place one test mouse in a fresh testing chamber and place the chamber within the enclosed testing area for 30 min. (This habituation period will not be analyzed, so video recording during this period is optional.)
    3. Separate stimulus mice to individual clean cages kept outside of the enclosed area for 30 min.
    4. Following the test mouse’s 30-min habituation to the testing chamber, place an inverted wire cup in the middle of the right half of the testing chamber (Figure 3). The stimulus mouse should be able to explore the entire cup perimeter.
    5. Allow the mouse to explore the cup for 10 min.
      Note: This exploration period will not be analyzed, so video recording during this period is optional.

      Figure 3. Placement of stimulus cup with testing apparatus

  4. Habituation test trials (Trials 1-8)
    1. Begin video recording.
    2. Lift one side of the cup and gently place stimulus mouse #1 underneath (Figure 4). Complete placement underneath the cup within 10 s.

      Figure 4. Placement of stimulus mouse under wired cup

    3. Immediately start a stopwatch or use a clock to time 1 min. Note the time of day in a lab notebook. An example worksheet that can be used for this purpose is provided as Supplemental File 1.
    4. After 1 min, quickly remove the stimulus mouse from the wire cup and place back in its individual cage.
    5. Allow the test mouse to explore the testing chamber and empty wire cup for the remaining 9 min. This marks the end of the first trial.
    6. Repeat Steps D1-D5 until 8 trials have been completed, using stimulus mouse #1 for each trial.

  5. Dishabituation test trial (Trial 9)
    1. After completion of the 8th trial, repeat Steps D2-D4 using stimulus mouse #2 (Figure 5). This marks the end of the 9th trial and the end of testing.
      Note: If testing another mouse immediately after, clean the wire cup and the sides of the cage with 70% EtOH followed by DI Water, wipe dry with a paper towel, and replace the bedding. When testing for a given day is completed, stimulus mice can be returned together to their home cage, if housed together prior to testing.

      Figure 5. Timeline of protocol

  6. Video Coding for Blinded Analysis
    1. Each 90-min testing period is recorded as 9 separate 10-min video recordings.
    2. Create an Excel spreadsheet (Figure 6A).
    3. In the first column (e.g., Column A), type the original names for the files (e.g., CageId-Trial#), such that each row represents one trial.
    4. In the second column (e.g., Column B), type the following code into the function tab above the Excel cells: =CHAR(RANDBETWEEN(65,90))andCHAR(RANDBETWEEN(65,90))andRANDBETWEEN(10,99)
    5. Use the corner of the bottom right of the cell and drag it down to assign that function to all the rows to create a key.
    6. Highlight the codes and copy them. Right-click in the first cell of the column to the right (e.g., Column C), select “Paste Special,” and select “Paste Values” (Figure 6B). (If “Paste Values” is not used, the values generated by the code will continue to change anytime a change is made in the spreadsheet.)
    7. Rename the files according to the corresponding codes. Sort files alphabetically by file name to shuffle the files out of order (Figure 6C). Follow the alphabetical order of coded file names during analysis.
      Note: When renaming files for blinded analysis, make sure to have an alternate way of identifying the video. For example, videos recorded in iSpy have the time in the corner of the image, which can serve as back-up identification. When running the behavioral tests, note the times of each trial in a notebook in case a file is misnamed.
    8. Once blinded analysis is complete, refer to the key created In the Excel sheet to match the code-named file to the respective mouse and trial.

      Figure 6. Example of random file naming/coding on Excel and sample blinding file. A. The “key” is created in Excel by listing the original file names in Column A. The function is added to the adjacent column to produce the randomized “blinding” names. B. After copying the names and selecting “Paste Special: Values”, the key is complete and will not change. From there, the respective file names are renamed with this randomized 4-digit name. C. Finally, the files are sorted alphabetically by 4-digit name. When scoring the videos, follow this alphabetized order to shuffle the videos for blinded analyses.

Data analysis

Quantification of social interaction
Social interaction can be quantified with a stopwatch. Interaction is defined as direct contact of the test mouse’s nose to the cup or stimulus mouse, or position of the test mouse’s nose within 1 cm of the wire cup, with the nose facing the stimulus mouse (Figure 7). Social interaction is analyzed from the time the cage top is placed on the cage to 1 min after. Evaluate and score 1 full minute with a stopwatch for each trial. Plot the data as shown in Figure 8A.

Figure 7. Mouse engaging in “social interaction”. In the image on the left, the test mouse is away from the cup and its nose is not within 1 cm of it. This behavior would not be counted as sniffing time. In the image on the right, the test mouse’s nose is making direct contact with the cup and facing toward the cup; this behavior is thus included in the sniffing time.

Figure 8. Example data from a habituation/dishabituation test. A. The social interest (sniffing time) of the test mouse (n = 1) decreases over the first 8 trials and increases for the 9th trial when the novel mouse is introduced. B. Repetitive self-grooming times in the 1st, 4th, and 8th trials. This example illustrates representative Dbi+/+ and Dbi-/- mice (n = 1 each). Full comparisons between these genotypes are presented in Ujjainwala et al., 2018.

Repetitive self-grooming
Repetitive self-grooming can also be scored with a stopwatch using the portions of videos covering min 2-10 of each trial. Repetitive self-grooming is defined as a period of at least 10 s spent grooming, with no more than 5 s between consecutive grooming spurts (Silverman et al., 2010). For an initial analysis, record self-grooming times for min 2-10 of trials 1, 4, and 8 to assess grooming at the beginning, middle, and end of testing. Plot the data as shown in Figure 8B. The remaining trials can be analyzed for repetitive grooming time if more comprehensive quantification is desired. If removing the stimulus mouse from the testing cage is not done consistently and rapidly for every trial, a different interval, such as min 3-10, may be used.

Statistical analysis

  1. Enter data into SAS or other statistics software.
  2. Analyze sniffing time and repetitive grooming time (in s) using repeated measures analysis of variance (ANOVA).
  3. Enter trial as within-subjects factor, and enter sex and genotype as between-subjects factors.
  4. To test for differences in social interest, compare sniffing time (in s) across trials 1-9 to test for significance. In addition, compare the total sniffing time summed across trials 1-8 to test for significant differences in total levels of interest displayed toward the first stimulus mouse.
  5. To test for differences in social recognition, compare sniffing time (in s) across trials 8 and 9 to test for significance.
  6. To test for differences in repetitive self-grooming, compare repetitive grooming time (in s) across the intertrial intervals for which grooming was quantified.


  1. Unless explicitly studying the effects of social isolation, all test mice should be group-housed until testing, as social isolation can lead to altered social behavior (Lander et al., 2017).
  2. Tests should be conducted at a consistent time of day. For example, our lab routinely performs these tests between 9:00 AM and 1:00 PM (relative to 7:00 PM lights off).
  3. If possible, stimulus mice and test mice should be housed in different animal facility rooms. If that option is not available, physically separate the cages housing stimulus and test mice on a cage rack with at least one row in between. If the animal facility uses fully enclosed individually ventilated cages (IVCs), thus minimizing olfactory and pheromonal signals, separation such that there is no visual interaction is suggested.
  4. The first time the stimulus mouse is placed under the cup should not be during an experiment. Stimulus mice tend to exhibit behaviors reflecting anxiety, such as clinging to wires of the cup or cage-biting, in response to initial exposure to the cup. Train the stimulus mice by using non-test mice prior to running experimental mice.
  5. When transporting mice (test or stimulus) between cages, minimize the time the mouse is moved without having contact with a support surface underneath as hanging the mouse for significant time is a stressor for mice (Deacon, 2006). This support can be provided by placing the mouse on the sleeve of the investigator’s forearm covered by a clean lab coat, or placing on top of a cage lid with light restraint on the tail.
  6. In our studies, most mice continued to show habituation (as indicated by continued decreases in sniffing time) throughout the first 8 trials. However, if the test mice show stronger habituation earlier on, the number of trials prior to the “dishabituation phase” can potentially be reduced.
  7. If the test mouse is spending a significant amount of time in a part of the cage obscured by the wire cup, consider positioning the camera to record from a top-down view.
  8. Cross-validation of phenotypes across multiple behavioral assays can help solidify conclusions regarding the effect of a genetic mutation or other manipulation. Therefore, it may be advisable to test mice on another assay of social interest, such as the 3-chamber test (Yang et al., 2011), in addition to the social habituation/dishabituation test presented here.


We thank Dr. Emilie Rissman for helpful advice regarding the social habituation/dishabituation test procedures. This work was supported by the Brain and Behavior Research Foundation (NARSAD Young Investigator Grant 24086, C.A.C.). C.A.C. is also supported by NIH/NINDS grants R01 NS105825 and R03 NS103029. This protocol is adapted from Ujjainwala et al., 2018.

Competing interests

The authors declare no competing interests.


All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign (17237).


  1. Austin, C. P., Battey, J. F., Bradley, A., Bucan, M., Capecchi, M., Collins, F. S., Dove, W. F., Duyk, G., Dymecki, S., Eppig, J. T., Grieder, F. B., Heintz, N., Hicks, G., Insel, T. R., Joyner, A., Koller, B. H., Lloyd, K. C., Magnuson, T., Moore, M. W., Nagy, A., Pollock, J. D., Roses, A. D., Sands, A. T., Seed, B., Skarnes, W. C., Snoddy, J., Soriano, P., Stewart, D. J., Stewart, F., Stillman, B., Varmus, H., Varticovski, L., Verma, I. M., Vogt, T. F., von Melchner, H., Witkowski, J., Woychik, R. P., Wurst, W., Yancopoulos, G. D., Young, S. G. and Zambrowicz, B. (2004). The knockout mouse project. Nat Genet 36(9): 921-924.
  2. Crawley, J. N. (2007). Mouse behavioral assays relevant to the symptoms of autism. Brain Pathol 17(4): 448-459.
  3. Cryan, J. F. and Holmes, A. (2005). The ascent of mouse: advances in modelling human depression and anxiety. Nat Rev Drug Discov 4(9): 775-790.
  4. Deacon, R. M. (2006). Housing, husbandry and handling of rodents for behavioral experiments. Nat Protoc 1(2): 936-946.
  5. Ferguson, J. N., Young, L. J., Hearn, E. F., Matzuk, M. M., Insel, T. R., and Winslow, J. T. (2000). Social amnesia in mice lacking the OXT gene. Nat Genet 25(3): 284-288.
  6. Goldstein, B., and Rosselli, F. (2003). Etiological paradigms of depression: The relationship between perceived causes, empowerment, treatment preferences, and stigma. J Ment Heal12(6), 551-563.
  7. Karlsson, S. A., Haziri, K., Hansson, E., Kettunen, P., and Westberg, L. (2015). Effects of sex and gonadectomy on social investigation and social recognition in mice. BMC Neurosci 16: 83.
  8. Lander, S. S., Linder-Shacham, D. and Gaisler-Salomon, I. (2017). Differential effects of social isolation in adolescent and adult mice on behavior and cortical gene expression. Behav Brain Res 316: 245-254.
  9. Lewis, M. and Kim, S. J. (2009). The pathophysiology of restricted repetitive behavior. J Neurodev Disord 1(2): 114-132.
  10. McFarlane, H. G., Kusek, G. K., Yang, M., Phoenix, J. L., Bolivar, V. J. and Crawley, J. N. (2008). Autism-like behavioral phenotypes in BTBR T+tf/J mice. Genes Brain Behav 7(2): 152-163.
  11. Morato, S. and Castrechini, P. (1989). Effects of floor surface and environmental illumination on exploratory activity in the elevated plus-maze. Braz J Med Biol Res 22(6): 707-710.
  12. Saunders, S. A., and Roy, C. (1999). The relationship between depression, satisfaction with life, and social interest. South Pacific J Psychology 11(1): 9-15.
  13. Silverman, J. L., Yang, M., Lord, C. and Crawley, J. N. (2010). Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci 11(7): 490-502.
  14. Stevenson, E. L., and Caldwell, H. K. (2014). Lesions to the CA 2 region of the hippocampus impair social memory in mice. Eur J Neurosci 40(9): 3294-3301.
  15. Tejada, L. D., and Rissman, E. F. (2012). Sex differences in social investigation: effects of androgen receptors, hormones and test partner. J Neuroendocrinol 24(8): 1144-1153.
  16. Ujjainwala, A. L., Courtney, C. D., Rhoads, S. G., Rhodes, J. S., and Christian, C. A. (2018). Genetic loss of diazepam binding inhibitor in mice impairs social interest. Genes Brain Behav e12442.
  17. Wolstenholme, J. T., Edwards, M., Shetty, S. R. J., Gatewood, J. D., Taylor, J. A., Rissman, E. F., and Connelly, J. J. (2012). Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression. Endocrinology 153(8): 3828-3838.
  18. White, S. W., Oswald, D., Ollendick, T., and Scahill, L. (2009). Anxiety in children and adolescents with autism spectrum disorders. Clin Psychol Rev 29(3): 216-229.
  19. Yang, M., Silverman, J. L., and Crawley, J. N. (2011). Automated three‐chambered social approach task for mice. Curr Protoc Neurosci 56(1): 8-26.


[摘要 ] 识别同一物种成员并与之互动的能力对于社会交流至关重要。调查社会兴趣和认可的神经基础可能会提供洞察力,以了解影响社会行为的疾病中的行为差异。用于研究啮齿动物的社会兴趣的分析方法包括3室测试,分区测试和社交互动测试。在这里,我们提出了一个单一的协议,可用于量化小鼠显示的社会兴趣水平,区分不同个体小鼠的能力(社会识别)以及显示的重复自我修饰水平。在协议的第一部分中,进行了一项社会习惯/适应测试,即在9个试验中对一只测试小鼠嗅探刺激小鼠所花费的时间进行了量化。在前8次互动中,重复使用相同的刺激鼠标。在第九次试验中,提出了一种新型刺激小鼠。通过在试验1-8中逐渐减少调查时间,在试验9中增加,可以表示完整的社会认可。每次社会试验之间的间隔用于量化自我修饰,这是小鼠的定型重复行为。我们还提出了一种对这些行为进行随机,盲法分析的方法,以提高结果的严谨性和可重复性。因此,这种单一的行为测试能够以一种综合的方式在同一只动物中容易地评估社交和重复行为的表型。此功能在理解具有自闭症和其他神经发育或神经精神疾病相关的遗传变异的小鼠模型中这些行为和表型之间的相互作用时可能是有利的,这些变异通常以这些行为差异为特征。
[背景 ] 言语和非言语沟通增强生物体的建立和维护一个社会阶层,因此生存和繁殖能力。这些形式的交流依赖与同伴互动的动机以及识别和区分不同个体的能力。在人类中,通常以社交行为改变为特征的疾病包括抑郁症,焦虑症和自闭症(Saunders和Roy,1999; White 等人,2009)。尽管人们对症状有一个普遍的了解,但这些疾病的病因,尤其是社会障碍和差异的潜在机制仍不清楚(Goldstein and Rosselli ,2003)。

小鼠提供有利模型小号(奥斯汀通过类似人类的行为和生理的某些方面的学习行为的神经生物学等人。,2004年Cryan 和霍姆斯,2005)。铝虽然小鼠模型不会完美地复制人类疾病,症状总能调查有关遗传或环境调制(克劳利,2007)的理论时适用。例如,当前的许多临床前自闭症研究都围绕异常社交互动,社交交流和互动受损以及重复行为的诊断标准(Silverman 等,2010)。

通过社交习惯/适应测试,可以测量啮齿动物社交行为的两个组成部分:1)对在短时间内反复显示的熟悉的社会刺激的兴趣;2)认识到新的社会刺激的能力(Tejada和Rissman ,2012;Ujjainwala 等,2018)。在此范例中,将用于提供社交刺激的鼠标放在金属杯下,以将其限制在固定位置,并将其与正在检查其行为的鼠标(“测试鼠标”)物理隔离。兴趣是通过测试老鼠花在铁丝罩下探索刺激老鼠的时间量来衡量的,通常通过嗅探来量化。该范例可用于调查对社会兴趣和/或社会认可的生物和环境影响。例如,我们的研究小组最近报告说,与野生型同窝仔相比,遗传上缺乏地西epa结合抑制剂(DBI)的小鼠显示出明显的社会兴趣降低,而社交识别能力没有下降(Ujjainwala 等人,2018)。此外,啮齿动物通常在社会兴趣水平上表现出性别差异(Tejada和Rissman ,2012; Karlsson 等,2015),而性甾体激素(例如睾丸激素和雌二醇)会影响社会探索行为。此的变化协议也已经被用于检查所述的损失ø xytocin基因以及病变到的CA2区域ħ ippocampus 及其对社会识别效果(弗格森等人,2000; 史蒂文森和考德威尔,2014 )。此外,该测试可应用于进一步评估的其他环境和行为因素的影响,如社会隔离或妊娠暴露于影响b isphenol A(五星行等人,2012 ; 兰德。等人,201 7 )。

限制性重复行为最常与人类自闭症相关,但这些行为也存在于其他疾病中,例如Rett,Fragile X和Prader-Willi综合征(Lewis和Kim,2009; Crawley,2007; McFarlane 等,2008)。 )。在小鼠中,该表型可以多种形式出现,包括重复修饰,盘旋,跳跃或其他定型运动(Crawley,2007)。鉴于这些和​​其他神经精神病学和神经发育障碍中社交互动受损和重复行为之间存在重叠,因此能够对多种表型进行现成和综合评估的单一方案是有利的。

通常用于研究小鼠的社会兴趣和识别的另一范式是3室测试。该测试需要一个专门的设备,该设备包含多个由可移动门隔开的腔室。与镜像家笼的环境相比,这种配置对老鼠具有更高的新颖性,有可能导致更高水平的一般探索行为,而不是特定的社会互动(Yang 等,2011)。相反,在此协议中描述的测试能够进行在许多实验室用于日常壳体中的标准鼠笼。可以以低成本容易地获得所需的其他材料。


关键字:行为, 习惯化, 去习惯化, 理毛, 盲态分析, 社交兴趣, 社会识别



铬圆形笔筒(频谱多样化,目录Ñ 棕土:31570,10.2厘米直径的,为0.64cm杆之间的距离,的10.8厘米初始高度)
注意:对于此过程,至少需要两只刺激小鼠。确保两只刺激小鼠的性别和年龄相同,并且都没有与任何测试小鼠接触(即没有同窝仔)。刺激小鼠可以是雄性或雌性,因为它们在用杯子进行测试的过程中被物理隔离。通常,女性卵巢切除用于提供中性stimul 我来量化不会引发攻击性或性反应社会利益。如有必要,请咨询当地动物保健委员会有关卵巢切除手术的指南。




MiliporeSigma Synergy超纯水净化系统(MiliporeSigma ,型号:SYNSOHFUS)
Windows PC电脑
摄像机(Logitech Webcam C930e,90 ° 视野,1920 x 1080像素)

测试室(可以使用标准鼠标笼的底部和过滤器顶部,底部的尺寸为29.3 cm x 17.1 cm x 12.7 cm,盖的顶部总高度为20.3 cm)



Microsoft Excel



注意:如果金属丝杯不够高,无法防止鼠标爬到顶部,请用胶水或胶带将400 ml塑料烧杯的底部固定在金属丝杯的底部,以延长金属丝杯的高度,如图的图1的总体高度杯> 14厘米应PREV ë 从栖息在杯的顶部NT最小鼠。





在测试区域内,灯光应昏暗,正上方不应有任何强光,因为光线水平会影响小鼠的探索行为(Morato 和Castrechini ,1989 )。使用光电平(pproximately 3 - 6勒克司)足以以显现整个保持架和测试小鼠通过视频摄像机。这种照明可以通过间接光来实现。

将相机连接到视频软件iSpy 。
编辑相机设置,将记录时间设置为610 s(10分钟加10 s),最大帧速率设置为10 FPS,缓冲时间设置为2 s。每个试用期为10分钟;另外的10 s用于将刺激鼠标放置在杯子中。

D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图2.jpg



将刺激物和测试小鼠带到各自笼中的测试室。运输过程中,笼子应分别用不透明的布或深色塑料袋覆盖。将小鼠关在笼子里,直到准备好让它们适应测试室。放置刺激小鼠和试验小鼠的笼子应至少保持1.5 m的距离,以防止试验前发生任何相互作用。


D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图3.jpg



提起杯子的一侧,然后将刺激鼠标1轻轻放在下面(图4 )。在10秒钟内将其完全放置在杯子下方。

D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图4.jpg

图4 。将刺激鼠标放在有线杯下


1 分钟后,快速从电线杯中取出刺激鼠标,然后放回其单独的笼子中。
重复步骤D 1- D 5,直到完成8个试验为止,每个试验都使用刺激鼠标#1。

在完成第8 个试验后,使用2号刺激鼠标重复图D2-D 4 (图5 )。这标志着第9 次审判的结束和测试的结束。


D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \ 5.jpg

图5 。协议时间表


在第一列(例如,列A)中,键入文件的原始名称(例如,CageId -Trial#),以使每一行代表一个试验。
在第二列(例如,列B)中,在Excel单元上方的功能选项卡中键入以下代码:= CHAR(RANDBETWEEN(65,90))和CHAR(RANDBETWEEN(65,90))和RANDBETWEEN(10,99)
突出显示代码并复制它们。右键单击右侧列的第一个单元格(例如C列),选择“ Paste Special”,然后选择“ Paste Values”(图6B)。(如果未使用“粘贴值”,则每次在电子表格中进行更改时,由代码生成的值将继续更改。)
注意:重命名文件以进行盲法分析时,请确保使用其他方法来识别视频。例如,以iSpy 录制的视频的时间在图像的一角,可以用作备份标识。运行行为测试时,请记下笔记本中每次试用的时间,以防文件名错误。

D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图6-MS.jpg

图6 。在Excel和示例盲文件上随机文件命名/编码的示例。答:“密钥”是通过在列A中列出原始文件名在Excel中创建的。该函数被添加到相邻列中以产生随机的“盲”名。B.复制名称并选择“特殊粘贴:值”后,密钥已完成并且不会更改。从那里开始,各个文件名都用这个随机的4位数字名重命名。C.最后,文件按4位数字字母顺序排序。对视频评分时,请按照字母顺序对视频进行混洗以进行盲目分析。







D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图7.jpg

图7.鼠标参与“社交互动” 。在左图中,测试鼠标远离杯子,并且其鼻子不在杯子的1厘米之内。此行为将不被视为嗅探时间。在右图中,测试鼠标的鼻子直接与杯子接触,并面向杯子;因此,该行为包括在嗅探时间中。


D:\ Reformatting \ 2019-12-16 \ 1902770--1258 Catherine Christian 810605 \ Figs jpg \图8-MS.jpg

图8.来自适应/适应测试的示例数据。答:在引入新小鼠时,测试小鼠(n = 1)的社会兴趣(嗅觉时间)在前8次试验中降低,而在第9 次试验中增加。B.在第一次,第四次和第八次试验中重复自我修饰的时间。此示例说明了代表性的Dbi + / + 和Dbi -/- 小鼠(每个n = 1)。这些基因型之间的全面比较在Ujjainwala et al 。,2018中进行了介绍。



重复的自我修饰也可以使用秒表对每次试验中至少2-10分钟的视频部分进行评分。重复自我修饰被定义为至少10秒的修饰时间,两次连续修饰之间的间隔不超过5秒(Silverman 等,2010)。为了进行初步分析,请记录试验1,试验4和试验8的2-10分钟的自我修饰时间,以评估测试开始,中期和结束时的修饰情况。绘制数据,如图8B所示。将R emaining试验,如果能更全面的量化需要进行分析梳理重复一次。如果对于每个试验而言,从测试笼子中取出刺激性老鼠的操作均不能始终如一地迅速进行,则可以使用不同的间隔,例如最小3-10分钟。



将数据输入SAS 或其他统计软件。
使用重复测量方差分析(ANOVA)分析嗅探时间和重复修饰时间(以s 为单位)。
要测试社会兴趣方面的差异,请比较试验1-9 中的嗅探时间(s ),以检验其重要性。此外,比较试验1-8中汇总的总嗅探时间,以测试向第一只刺激小鼠显示的总关注水平的显着差异。
要测试社会认可度的差异,请比较试验8和9 中的嗅探时间(s ),以检验其重要性。




除非明确研究社交隔离的影响,否则所有测试小鼠都应分组饲养直到进行测试,因为社交隔离会导致社交行为发生变化(Lander 等,2017 )。
测试应在一天中的一致时间进行。例如,我们的实验室通常在9:00 AM和1:00 PM之间执行这些测试(相对于7:00 PM熄灭)。
跨多种行为分析对表型进行交叉验证可以帮助巩固有关基因突变或其他操作效果的结论。因此,除了此处介绍的社交习惯/适应能力测试外,建议对小鼠进行另一项具有社会意义的检测方法,例如三室测试(Yang 等,2011)。



我们感谢埃米莉· 里斯曼(Emilie Rissman)博士对社交习惯/适应测试程序的有用建议。这项工作得到了脑与行为研究基金会(NARSAD Young Investigator Grant 24086,CAC)的支持。NIH / NINDS赠款R01 NS105825和R03 NS103029也支持CAC。该协议改编自Ujjainwala 等人,2018。











奥斯汀,CP,Battey ,JF,布拉德利,A.,Bucan ,M.,卡佩奇,M.,柯林斯,FS,多芬,WF,Duyk ,G.,Dymecki ,S.,Eppig ,JT,Grieder ,FB,海因茨,N.,Hicks,G.,Insel ,TR,Joyner,A.,Koller,BH,Lloyd,KC,Magnuson,T.,Moore,MW,Nagy,A.,Pollock,JD,Roses,AD,Sands, AT,Seed,B.,Skarnes ,WC,Snoddy ,J.,Soriano,P.,Stewart,DJ,Stewart,F.,Stillman,B.,Varmus,H.,Varticovski ,L.,Verma,IM,Vogt ,TF,von Melchner ,H.,Witkowski,J.,Woychik ,RP,Wurst ,W.,Yancopoulos ,GD,Young,SG和Zambrowicz ,B。(2004)。敲除鼠标项目。Nat Genet 36(9):921-924。
Cryan ,JF和Holmes,A。(2005)。鼠标的上升:在模拟人的抑郁和焦虑方面的进展。Nat Rev Drug Discov 4(9):775-790。
迪肯(RM)执事(2006)。进行行为实验的啮齿动物的住房,饲养和处理。纳特Protoc 1(2):936-946。
弗格森(JF),扬(Young),LJ,赫恩(Hearn),英孚(EF),马祖克(MM),英瑟(Insel),TR和温斯洛(JT)温斯洛(2000)。缺乏OXT基因的小鼠的社会失忆症。Nat Genet 25(3):284-288 。
Goldstein,B.和Rosselli ,F.(2003)。抑郁的病因学范式:感知原因,授权,治疗偏好和污名之间的关系。Ĵ 精神疾病Heal12 (6),551-563。
南卡罗来纳州卡尔森(Karlsson),哈齐里(Haziri ),汉森(Hansson),凯特顿(Kettunen)和西湖(Westberg)(2015)。性别和性腺切除术对小鼠的社会调查和社会认可的影响。BMC Ñ eurosci 16 :83。
兰德,SS,Linder- 沙哈姆,D和Gaisler -Salomon,I.(2017年)。社会隔离对青春期和成年小鼠行为和皮质基因表达的差异作用。Behav 脑水库316:245 - 254 。
Lewis,M.和Kim,SJ(2009)。限制性重复行为的病理生理学。Ĵ Neurodev Disord 1(2):114-132。
McFarlane,HG,Kusek ,GK,Yang,M.,Phoenix,JL,Bolivar,VJ和Crawley,JN(2008)。BTBR T + tf / J小鼠中的自闭症样行为表型。基因脑Behav 7(2):152-163。
Morato ,S。和Castrechini ,P。(1989)。地板表面和环境照明对高架迷宫中探索活动的影响。Braz J Med Biol Res 22(6):707-710。
Saunders,SA和Roy,C.(1999)。抑郁,对生活的满意度和社会兴趣之间的关系。南太平洋J心理学11 (1):9-15。
Silverman,JL,Yang,M.,Lord C. and Crawley,JN(2010)。自闭症小鼠模型的行为表型分析。Nat Rev Neurosci 11(7):490-502。
EL,史蒂文森和香港考德威尔(2014)。海马CA 2区的病变损害小鼠的社交记忆。Eur J Neurosci 40 (9):3294-3301。
特哈达(LD)和里斯曼(EF)(2012)。社会调查中的性别差异:雄激素受体,激素和测试对象的影响。Ĵ Neuroendocrinol 24 (8) :1144至1153年。
Ujjainwala ,AL,Courtney,CD,Rhoads,SG,Rhodes,JS和Christian,CA(2018)。地西epa结合抑制剂在小鼠中的遗传损失损害了社会利益。基因脑Behav e12442。
Wolstenholme,JT,Edwards,M.,Shetty,SRJ,Gatewood,JD,Taylor,JA,Rissman ,EF和Connelly,JJ(2012)。妊娠期暴露于双酚a会导致行为和基因表达发生跨代变化。内分泌学153(8):3828-3838。
White,SW,Oswald,D.,Ollendick ,T。,和Scahill ,L。(2009)。自闭症谱系障碍儿童和青少年的焦虑症。临床心理学版本29(3) :216 - 229。
Yang,M.,Silverman,JL和Crawley,JN(2011)。自动化的3 - 腔社会的方法任务小鼠。Curr Protoc Neurosci 56 (1):8-26。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lawande, N. V., Ujjainwala, A. L. and Christian, C. A. (2020). A Single Test to Study Social Behavior and Repetitive Self-grooming in Mice. Bio-protocol 10(2): e3499. DOI: 10.21769/BioProtoc.3499.

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。