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Social recognition memory is essential for the establishment and maintenance of a rodent colony. Recognition memory is important for social hierarchy, mate and offspring recognition, and interspecies recognition. Interspecies recognition is vital for recognizing frequent visitors to the animal’s habitat and whether or not the visitors pose a threat to the animals or colony (Macbeth et al., 2009; Noack et al., 2010). Here, we describe a protocol which effectively and reproducibly measures the social recognition for a juvenile male, a female, a mouse of another strain, and a rat. This task relies on the animal’s innate tendency to explore a novel social partner and decrease the exploration of a known familiar social partner (Thor et al., 1982). A significant decrease in the exploration of a partner from the training session to the recall session demonstrates a memory of the social partner. Also, we describe a social recognition procedure, the habituation-dishabituation paradigm that closely mimics typical short, frequent interactions between animals in a colony (Dantzer et al., 1987; Winslow and Camacho, 1995). Further, olfaction is a key component of social recognition, to test olfaction see Jacobs et al. (2016). In this protocol, we use transgenic NR2A overexpression mice to demonstrate how an impairment in social recognition memory may appear.

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Social Recognition Memory Test in Rodents
啮齿动物社会认知记忆测试

神经科学 > 行为神经科学 > 学习和记忆
作者: Stephanie A. Jacobs
Stephanie A. JacobsAffiliation: Department of Neurology, Brain and Behavior Discovery Institute, Medical College of Georgia, Augusta University, Augusta, USA
For correspondence: stjacobs@augusta.edu
Bio-protocol author page: a3112
Fengying Huang
Fengying HuangAffiliation: Department of Neurology, Brain and Behavior Discovery Institute, Medical College of Georgia, Augusta University, Augusta, USA
Bio-protocol author page: a3113
Joe Z. Tsien
Joe Z. TsienAffiliation: Department of Neurology, Brain and Behavior Discovery Institute, Medical College of Georgia, Augusta University, Augusta, USA
Bio-protocol author page: a3114
 and Wei Wei
Wei WeiAffiliation: Banna Biomedical Research Institute, Xi-Shuang-Ban-Na Prefecture, Xishuangbanna, China
For correspondence: wwei@bbri.ac.cn
Bio-protocol author page: a3115
Vol 6, Iss 9, 5/5/2016, 1937 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1804

[Abstract] Social recognition memory is essential for the establishment and maintenance of a rodent colony. Recognition memory is important for social hierarchy, mate and offspring recognition, and interspecies recognition. Interspecies recognition is vital for recognizing frequent visitors to the animal’s habitat and whether or not the visitors pose a threat to the animals or colony (Macbeth et al., 2009; Noack et al., 2010). Here, we describe a protocol which effectively and reproducibly measures the social recognition for a juvenile male, a female, a mouse of another strain, and a rat. This task relies on the animal’s innate tendency to explore a novel social partner and decrease the exploration of a known familiar social partner (Thor et al., 1982). A significant decrease in the exploration of a partner from the training session to the recall session demonstrates a memory of the social partner. Also, we describe a social recognition procedure, the habituation-dishabituation paradigm that closely mimics typical short, frequent interactions between animals in a colony (Dantzer et al., 1987; Winslow and Camacho, 1995). Further, olfaction is a key component of social recognition, to test olfaction see Jacobs et al. (2016). In this protocol, we use transgenic NR2A overexpression mice to demonstrate how an impairment in social recognition memory may appear.

[Abstract]

Materials and Reagents

  1. Adult (3-8 months old) transgenic, knock out or treatment group mice (we use NR2A transgenic mice) and wild type mice (referred to as “subject mouse”)
  2. Juvenile (approximately 6-week old) male mice on the same genetic background
  3. Juvenile male mice on a different genetic background of a different color
  4. Approximately 3-month old adult female mice on the same genetic background
  5. Juvenile male rats
  6. 70% ethanol solution

Equipment

  1. Clean mouse cage identical to the home cage (standard mouse cage dimensions are 11 in x 7.5 in x 5 in), one for each subject mouse tested, change between sessions
  2. Clean rat home cage (standard rat housing cage dimensions are 22 in x 12.5 in x 8 in), one for each subject rat tested, change between sessions
  3. Clear Plexiglas to cover the cage top
  4. Small wire enclosure (for a female mouse), small wire pencil cup can be used (see Figure 1)
  5. Large wire mesh enclosure (for a juvenile male rat), made in-house from Plexiglas and wire mesh (Thermo Fisher Scientific, catalog number: AA42777HZ ) (see Figure 2)
  6. Digital recording camera
  7. Laboratory timer
  8. Stopwatch for recording investigation times

Procedure

  1. Experimental set up
    1. Experimental space should be quiet and dimly lit to avoid distraction from sounds and increased anxiety level from bright lights.
    2. The experimental area is surrounded by a back curtain with the experimenter outside of the curtain to avoid distraction from movements.
    3. A digital recording camera is mounted overhead and connected to a computer for recording the experiment.
    4. Social recognition experiments have two phases, a training phase and a recall phase. If the subject mouse does not spend at least 25 sec investigating the stimulus animal in the training phase the subject animal should be excluded for that experiment (Kogan et al., 2000). Insufficient exploration time in the training phase may lead to inconclusive or incorrect results.
    5. The subject mouse is said to be exploring the stimulus if the subject mouse is facing the stimulus and within 1 cm of the stimulus or following closely (≤ 1 cm) behind (Kogan et al., 2000).

  2. Social recognition in mice

    Social recognition of a conspecific juvenile male, or a juvenile male of a different strain
    1. Training phase
      1. Subject mice (transgenic mice and their wildtype littermates) should be group housed (2-5 mice) for best results as single housing mouse can lead to decreased social recognition memory (Kogan et al., 2000).
      2. Place the subject mouse into a clean plastic cage identical to the home cage for 30 min prior to testing to allow the animal to habituate to the cage.
      3. Place a juvenile male mouse (the stimulus mouse) into the cage with the subject mouse for 5 min.
      4. Allow the mice to explore each other, but if the mice begin to fight they should be separated immediately to avoid injury.
      5. After 5 min, both mice are placed back into their home cage.
    2. Recall phase
      1. For the recall phase, split the subject mice into two groups. One group will be placed with a novel juvenile male and the second group will be placed with the same juvenile male as in the training phase.
      2. Place the subject mouse into a clean plastic cage identical to the home cage for 30 min prior to testing. Reusing the same cage can skew the result as the subject mouse may have left its scent in the bedding of the cage.
      3. Place the appropriate juvenile male stimulus mouse into the cage with the subject mouse for 5 min.
      4. Allow the subject mouse to explore the stimulus mouse, but if the mice begin to fight they should be separated immediately to avoid injury.
      5. After 5 min, both mice are placed back into their home cage.

    Social recognition of a conspecific female
    1. Training phase
      1. Place the female mouse into the round wire enclosure in a clean cage for ten min two times the day before testing to reduce stress and fear in the female mouse (for required equipment see Figure 1).


        Figure 1. Experimental set-up for the female social recognition task. A. For the female social recognition task, a small wire enclosure and an empty clean mouse cage are used. B. This is an enlarged picture of the small wire enclosure used for the female mice.

      2. Place the subject mouse into a clean plastic cage identical to the home cage with the small wire enclosure for 30 min prior to testing to allow the animal to habituate to the cage and the wire enclosure.
      3. Briefly, lift the wire enclosure and place a female mouse into the wire enclosure, and allow the subject mouse to explore the wire enclosure containing the female mouse for 5 min.
      4. After 5 min, place both mice back into their home cage.
    2. Recall phase
      1. For the recall phase, split the subject mice into two groups. One group will be placed with a novel female and the second group will be placed with the same female as in the training phase.
      2. Place the subject mouse into a clean cage, identical to the home cage, with the wire enclosure in it, for 30 min prior to testing.
      3. Briefly, lift the wire enclosure and place the appropriate female mouse into the wire enclosure, and allow the subject mouse to explore the wire enclosure containing the female mouse for 5 min.
      4. After 5 min, both mice are placed back into their respective home cages.

    Social recognition of a mouse for a rodent of another species
    1. Training phase
      1. Place the rat into the wire mesh enclosure in a clean rat cage for ten minutes two times the day before testing to reduce stress and fear in the rat (for required equipment see Figure 2).


        Figure 2. Experimental set-up for the rat social recognition task. A. For the rat social recognition task, a large wire enclosure and an empty clean rat cage are used. B. This is an enlarged picture of the large wire enclosure used for the rat.

      2. Place the subject mouse into a clean plastic rat cage with the wire mesh enclosure for 30 min prior to testing to allow the mouse to habituate to the cage and the wire enclosure.
      3. Place a rat securely into the wire mesh enclosure, and the enclosure into the cage with the adult mouse for 5 min.
      4. Monitor the rat and mouse pair to ensure that the rat is not able to escape the enclosure for the safety of the mouse.
      5. After 5 min, place the mouse and the rat back into their respective home cages.
    2. Recall phase
      1. For the recall phase, split the subject mice into two groups. One group will be placed with a novel rat (this is the control group) and the second group will be placed with the same rat as in the training phase (this is the experimental group).
      2. Place the subject mouse into a clean plastic rat cage with the wire enclosure in it, for 30 min prior to testing.
      3. Place the appropriate rat into the cage with the adult mouse for 5 min allowing the male mouse to explore the rat in its enclosure.
      4. After 5 min, both animals are placed back into their home cages.

  3. Habituation-dishabituation in mice
    This paradigm mimics the short, frequent interactions that mice may experience traveling throughout the colony (Dantzer et al., 1987; Winslow and Camacho, 1995). It consists of five short exploration sessions each with a one minute duration.
    1. Place the subject mouse into a clean plastic cage identical to the home cage for 30 min prior to testing to allow the animal to habituate to the cage.
    2. Place a novel juvenile male conspecific into the cage with the subject mouse for 1 min. After one minute remove the stimulus mouse and place it into a holding cage.
    3. After a ten-minute delay, place the same stimulus mouse back into the cage with the subject mouse. Repeat this two more times for a total of 4 exploratory sessions.
    4. After the fourth ten-minute delay, the fifth exploratory session, place a novel juvenile conspecific into the cage with the subject mouse. After one minute remove the stimulus mouse from the cage.

  4. Data analysis
    1. Using a stopwatch, record the amount of time that the subject mouse spends investigating the stimulus animal. Investigation of the stimulus animal is defined as the subject mouse being within one centimeter of the subject mouse with its head directed toward the other animal, touching or licking the other animal (Figure 3, Video 1). Recently, Arakawa et al. (2014) has also developed an automated software that can be used to detect social interaction patterns, if this method is preferred. The software was shown to produce similar results to human observation but may not be applicable in all instances (Arakawa et al., 2014).


      Figure 3. Data analysis for the social recognition task. This diagram illustrates the investigation criteria of the subject mouse. For both examples, the subject mouse must be within 1 cm of the stimulus animal with its face directed toward the stimulus. A. This panel illustrates an example of the subject mouse investigating the facial region of the stimulus mouse. B. This is a picture of the subject mouse investigating the facial region of the stimulus mouse. C. This panel illustrates an example of the subject mouse investigating the ano-genital region of the stimulus mouse. D. In this picture, the stimulus mouse is investigating the ano-genital region of the stimulus mouse.

      Video 1. Sample video clip for the social recognition task

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. Record the time in seconds.
    3. Group the animals according to the genotype, sex, or treatment.
    4. Average the exploration times and determine the standard error.
    5. Plot the data using the standard error measurements as the error bars.
    6. Differences between the training session and the recall session can be determined using a Student’s T-test. The animal is said to have formed a memory of the stimulus animal if there is a significant decrease in the amount of time spent investigating the familiar stimulus animal from the training session to the recall session. Differences among groups can be determined by an ANOVA.

Representative data

  1. Social recognition task
    Figure 4 is an example of representative data collected during a social recognition paradigm with a juvenile male conspecific with a 24-h delay. Figure 4A and 4B are representative data from the group matched with a novel animal in the recall phase; this is the control group of the experiment. The exploration times are recorded in seconds and each group is averaged together and the standard error is calculated. This data is then plotted. The subject animals are expected to spend a similar amount of time investigating the novel animals in the training phase, as well as the novel animal in the recall phase. This indicated that any decreases in exploration times with a familiar animal are due to a memory of the familiar animal. Figure 4C and 4D are representative data from the group paired with the identical mouse in the recall phase. The wild type mice in the example do significantly decrease the exploration of the familiar mouse in the recall phase, indicating a memory of the stimulus animal. This significant decrease indicates that the wildtype mice have formed a memory for the conspecific. The NR2A mice are known to have impaired social recognition memory and do not show a significant decrease in the exploration time of the familiar mouse in the recall phase after a 24-h delay (Jacobs and Tsien, 2014). Significance between the phases is determined by a Student’s T-test and differences among the groups are determined by an ANOVA test.


    Figure 4. Representative data for the male conspecific recognition. A. Representative exploration times, in seconds, for the juvenile male conspecific task for both the wildtype and the NR2A transgenic mice. The identical juvenile male conspecific was used in both sessions. B. Graphical representative of the averages of the exploration times with SEM error bars. C. Representative exploration times, in seconds, for the juvenile male conspecific task for both the wildtype and the NR2A transgenic mice. A novel juvenile male conspecific was presented in the recall session. D. Graphical representative of the averages of the exploration times with SEM error bars. A novel juvenile conspecific was presented in the recall session.

  2. Habituation-Dishabituation in mice
    Figure 5 is an example of representative data collected during a habituation-dishabituation paradigm of social recognition. In this paradigm, the subject mouse is introduced to a conspecific stimulus mouse four times for one minute each followed by a single one minute exploration session with a novel mouse. The final session acts as a control for the expected decrease in exploration of the familiar stimulus over the first four sessions. Figure 5A is a table listening representative data for all five trials. It is expected that the exploration times of trials one through four decreases as the stimulus conspecific becomes familiar to the subject mouse. The data for each exploration by each group is averaged and the standard error is calculated. Figure 5B is the average exploration for each group plotted by trial number. Significance between consecutive trials is determined by a Student’s T-test and differences among groups are determined by an ANOVA test.


    Figure 5. Representative data for the habituation-dishabituation paradigm. A. Representative exploration times, in seconds, for each of the habituation-dishabituation paradigm for both the wildtype and the NR2A transgenic mice. The identical juvenile male conspecific was used in the first four sessions with a novel male conspecific in the fifth session. B. Graphical representative of the averages of the exploration times with SEM error bars.

Notes

Although the described protocol is relatively simple to perform and involves little specialized equipment, proper execution and attention to detail is essential for correct results and reproducibility.

  1. Firstly, the experimental set up should be in a slightly darkened, quiet room as mice often show fear of brightly lit area and can be startled by sudden loud noises or movements (Liu et al., 2014).
  2. Similarly, the animals should be habituated to the empty cage and the empty chambers to avoid the animals displaying neophobia (Deacon, 2006). Do not reuse the same cage as feces and urine from the first partner maybe present in the bedding giving inconsistent or skewed results (Arakawa et al., 2008; Tolokh et al., 2013). Rat cages were used due to the size of the wire mesh enclosure needed for the rat. Be sure to habituate the subject mouse to this testing cage prior to the training protocol.
  3. The time duration between the train phase and the recall phase vary depending on the type of memory being tested. Typically for short-term memory the time duration is 1 hour or two hours. For long-term memory, typical time durations are 12-24 h.
  4. Stimulus animal selection is important for several reasons. Juvenile male mice are used to decrease the possibility of the adult subject mouse to exhibit aggressive behaviors (Hlinak and Krejci, 1991; Heinla et al., 2014). Mouse strains can differ in their social aggressiveness [i.e.: CD-1 mice (Golden et al., 2011), Tg2576 mice, an Alzheimer’s strain (Alexander et al., 2011), and mice with altered serotonin levels (Takahashi et al., 2012)] and more aggressive strains may need to be separated to avoid fighting (Levine et al., 1979; Serri and Ely, 1984). Additionally, mice that are singly housed, or have been used for breeding may be more aggressive. Fighting during the exploration trials can be dangerous for both the stimulus mouse and the subject mouse. If fighting is observed the trial should be stopped immediately and the animals separated to avoid injury.
  5. Social recognition may be diminished if the stimulus mouse is on a different strain (Macbeth et al., 2009; Jacobs and Tsien, 2012; Jacobs and Tsien, 2014). The females used as stimulus animals should be on the same genetic background due to the observation that female mice in the wild are more likely to breed with a male from the same genetic background (Macbeth et al., 2009).
  6. During the social recognition protocol described here, breeding was not to occur. The wire enclosure allows the male mouse to explore the scent of the female without mounting or mating behaviors.
  7. In the described protocol, a rat was used to investigate cross-species recognition. Juvenile male rats can be used and may be preferred due to the smaller size. The rat enclosure prevents the rat and mouse from have direct interaction as rats and prevent muricide by the rat (Noack et al., 2010). Be sure to thoroughly clean all enclosures between animals and sessions with 70% ethanol or a suitable alternative.

Acknowledgments

This work was supported by funds from the National Institute of Mental Health (MH060236), National Institute on Aging (AG024022, AG034663 & AG025918), USAMRA00002, and Georgia Research Alliance (all to JZT). This protocol was adapted from (Jacobs and Tsien, 2014; Jacobs et al., 2015).

References

  1. Alexander, G., Hanna, A., Serna, V., Younkin, L., Younkin, S. and Janus, C. (2011). Increased aggression in males in transgenic Tg2576 mouse model of Alzheimer's disease. Behav Brain Res 216(1): 77-83.
  2. Arakawa, H., Blanchard, D. C., Arakawa, K., Dunlap, C. and Blanchard, R. J. (2008). Scent marking behavior as an odorant communication in mice. Neurosci Biobehav Rev 32(7): 1236-1248.
  3. Arakawa, T., Tanave, A., Ikeuchi, S., Takahashi, A., Kakihara, S., Kimura, S., Sugimoto, H., Asada, N., Shiroishi, T., Tomihara, K., Tsuchiya, T. and Koide, T. (2014). A male-specific QTL for social interaction behavior in mice mapped with automated pattern detection by a hidden Markov model incorporated into newly developed freeware. J Neurosci Methods 234: 127-134.
  4. Dantzer, R., Bluthe, R. M., Koob, G. F. and Le Moal, M. (1987). Modulation of social memory in male rats by neurohypophyseal peptides. Psychopharmacology (Berl) 91(3): 363-368.
  5. Deacon, R. M. (2006). Housing, husbandry and handling of rodents for behavioral experiments. Nat Protoc 1(2): 936-946.
  6. Golden, S. A., Covington, H. E., 3rd, Berton, O. and Russo, S. J. (2011). A standardized protocol for repeated social defeat stress in mice. Nat Protoc 6(8): 1183-1191.
  7. Heinla, I., Leidmaa, E., Visnapuu, T., Philips, M. A. and Vasar, E. (2014). Enrichment and individual housing reinforce the differences in aggressiveness and amphetamine response in 129S6/SvEv and C57BL/6 strains. Behav Brain Res 267: 66-73.
  8. Hlinak, Z. and Krejci, I. (1991). Social recognition in male rats: age differences and modulation by MIF-I and Alaptide. Physiol Res 40(1): 59-67.
  9. Jacobs, S. A. and Tsien, J. Z. (2012). Genetic overexpression of NR2B subunit enhances social recognition memory for different strains and species. PLoS One 7(4): e36387.
  10. Jacobs, S. A. and Tsien, J. Z. (2014). Overexpression of the NR2A subunit in the forebrain impairs long-term social recognition and non-social olfactory memory. Genes Brain Behav 13(4): 376-384.
  11. Jacobs, S. A., Huang, F., Tsien, F. J. and Wei, W. (2016). Olfactory recognition memory test in mice. Bio-protocol 6(9): e1803.
  12. Jacobs, S., Wei, W., Wang, D. and Tsien, J. Z. (2015). Importance of the GluN2B carboxy-terminal domain for enhancement of social memories. Learn Mem 22(8): 401-410.
  13. Kogan, J. H., Frankland, P. W. and Silva, A. J. (2000). Long-term memory underlying hippocampus-dependent social recognition in mice. Hippocampus 10(1): 47-56.
  14. Levine, L., Grossfield, J. and Rockwell, R. F. (1979). Functional relationships between genotypes and environments in behavior. Effects of different kinds of early social experience on interstrain fighting in male mice. J Hered 70(5): 317-320.
  15. Liu, J., Wei, W., Kuang, H., Tsien, J. Z. and Zhao, F. (2014). Heart rate and heart rate variability assessment identifies individual differences in fear response magnitudes to earthquake, free fall, and air puff in mice. PLoS One 9(3): e93270.
  16. Macbeth, A. H., Lee, H. J., Edds, J. and Young, W. S., 3rd (2009). Oxytocin and the oxytocin receptor underlie intrastrain, but not interstrain, social recognition. Genes Brain Behav 8(5): 558-567.
  17. Noack, J., Richter, K., Laube, G., Haghgoo, H. A., Veh, R. W. and Engelmann, M. (2010). Different importance of the volatile and non-volatile fractions of an olfactory signature for individual social recognition in rats versus mice and short-term versus long-term memory. Neurobiol Learn Mem 94(4): 568-575.
  18. Serri, G. A. and Ely, D. L. (1984). A comparative study of aggression related changes in brain serotonin in CBA, C57BL, and DBA mice. Behav Brain Res 12(3): 283-289.
  19. Takahashi, A., Quadros, I. M., de Almeida, R. M. and Miczek, K. A. (2012). Behavioral and pharmacogenetics of aggressive behavior. Curr Top Behav Neurosci 12: 73-138.
  20. Thor, D. H., Wainwright, K. L. and Holloway, W. R. (1982). Persistence of attention to a novel conspecific: some developmental variables in laboratory rats. Dev Psychobiol 15(1): 1-8.
  21. Tolokh, II, Fu, X. and Holy, T. E. (2013). Reliable sex and strain discrimination in the mouse vomeronasal organ and accessory olfactory bulb. J Neurosci 33(34): 13903-13913.
  22. Winslow, J. T. and Camacho, F. (1995). Cholinergic modulation of a decrement in social investigation following repeated contacts between mice. Psychopharmacology (Berl) 121(2): 164-172.

材料和试剂

  1. 成年(3-8个月)转基因,敲除或治疗组小鼠(我们使用NR2A转基因小鼠)和野生型小鼠(称为"受试小鼠")
  2. 处于相同遗传背景的幼年(约6周龄)雄性小鼠
  3. 在不同遗传背景的不同颜色的幼年雄性小鼠
  4. 大约3个月大的成年女性小鼠在同一遗传背景
  5. 少年雄性大鼠
  6. 70%乙醇溶液

设备

  1. 清洁鼠笼与家笼相同(标准鼠笼尺寸为11英寸×7.5英寸×5英寸),每只测试的受试小鼠一只,在期间变化
  2. 清洁大鼠笼笼(标准鼠笼笼尺寸为22英寸×12.5英寸×8英寸),每个受试大鼠测试一次,在会话之间变化
  3. 清除有机玻璃覆盖笼顶
  4. 小线外壳(用于女性鼠标),可使用小线铅笔杯(见图1)
  5. 大型金属丝网罩(用于少年雄性大鼠),由Plexiglas和丝网(Thermo Fisher Scientific,目录号:AA42777HZ)内部制造(见图2)
  6. 数字录像机
  7. 实验室定时器
  8. 记录调查时间的秒表

程序

  1. 实验设置
    1. 实验空间应安静,昏暗,避免分心 从声音和增加的焦虑水平从明亮的灯光。
    2. 的 实验区被实验者的后幕包围 外面的窗帘,以避免分心的运动。
    3. 数字记录摄像机安装在顶部并连接到计算机用于记录实验
    4. 社会认知实验有两个阶段,一个训练阶段和   召回阶段。 如果主题鼠标不花费至少25秒 调查刺激动物在训练阶段的主体 动物应该被排除用于该实验(Kogan等人,2000)。在训练阶段的探索时间不足可能导致 不确定或不正确的结果
    5. 主题小鼠说 如果主题小鼠面对刺激,则探索刺激 并且在刺激的1cm内或紧随(≤1cm)后 (Kogan等人,2000)。

  2. 小鼠的社会认知

    对特定少年男性或不同品系的少年男性的社会认可
    1. 训练阶段
      1. 受试小鼠(转基因小鼠及其野生型 同窝动物)应该分组容纳(2-5只小鼠),以获得最佳结果 单只鼠标可以导致社会识别记忆减少 (Kogan等人,2000)。
      2. 将主题鼠标放在干净 塑料笼与家笼相同,在测试前30分钟 允许动物习惯于笼子。
      3. 将少年雄性小鼠(刺激小鼠)与主题小鼠笼子5分钟。
      4. 允许小鼠互相探讨,但如果小鼠开始战斗,他们应立即分离避免伤害。
      5. 5分钟后,将两只小鼠放回其笼中。
    2. 调用阶段
      1. 对于召回阶段,将受试小鼠分成两组。 一 组将放置一个新颖的少年男性和第二组 将与同样的少年男性放置在训练阶段。
      2. 将主题鼠标放入一个干净的塑料笼子里 家笼在测试前30分钟。 重复使用同一个笼子可能会扭曲 作为受试小鼠的结果可能已在床上用品中留下其香味 的笼子。
      3. 将适当的幼年雄性刺激小鼠放入笼子与主题小鼠5分钟。
      4. 允许主题小鼠探索刺激小鼠,但如果 老鼠开始战斗,他们应该立即分开避免 伤害。
      5. 5分钟后,将两只小鼠放回其笼中。

    对特定女性的社会认可
    1. 训练阶段
      1. 将雌性鼠标放入圆线 外壳在一个干净的笼子里十分钟,测试前一天两次 以减少女性小鼠的压力和恐惧(所需设备 参见图1)。


        图1.女性社交的实验设置 识别任务。 A.对于女性社会识别任务,一个小线   外壳和空的清洁鼠标笼。 B.这是一个 用于雌性小鼠的小线外壳的放大图片
      2. 将主题鼠标放入一个干净的塑料笼子里 家笼与小线外壳在测试前30分钟 允许动物习惯于笼子和线外壳。
      3. 简要地,提起电线外壳,并将一只雌性鼠标放入 电线外壳,并允许主题鼠标探索电线 包含雌性小鼠5分钟。
      4. 5分钟后,将两只小鼠放回自己的笼子里。
    2. 调用阶段
      1. 对于召回阶段,将受试小鼠分成两组。 一 组将放置一个小说女性和第二组将 放置与同样女性在训练阶段。
      2. 放置主题小鼠进入一个干净的笼子,相同的家笼,与 电线外壳,在测试前30分钟。
      3. 简而言之,抬起   电线外壳,并将适当的雌鼠放入电线   外壳,并允许主题鼠标探索电线外壳 含有雌性小鼠5分钟。
      4. 5分钟后,将两只小鼠放回它们各自的家笼中。

    其他物种的啮齿动物的鼠标的社会识别
    1. 训练阶段
      1. 将老鼠放入钢丝网罩中 清洁鼠笼10分钟,测试前一天两次 减少大鼠的压力和恐惧(所需设备参见图2)。


        图2.大鼠社会识别任务的实验设置。对于大鼠社会识别任务,大型金属丝外壳和 使用空清洁鼠笼。 B.这是一个放大的图片 用于老鼠的大线外壳
      2. 放置主题 小鼠进入一个干净的塑料鼠笼与线网罩30 在测试之前允许小鼠习惯于笼子和   电线外壳。
      3. 将老鼠牢固地放入金属丝网封套,并将笼子与成年老鼠笼子5分钟。
      4. 监测大鼠和小鼠对,以确保大鼠不能逃离外壳,为了鼠标的安全
      5. 5分钟后,将小鼠和大鼠放回他们各自的家笼。
    2. 调用阶段
      1. 对于召回阶段,将受试小鼠分成两组。 一 组将放置一只新型大鼠(这是对照组)和 第二组将与训练中的同一只大鼠放置 相(这是实验组)。
      2. 在测试前,将受试小鼠放入清洁的塑料大鼠笼子中,在其中放置电线外壳30分钟。
      3. 将适当的大鼠与成年小鼠笼中5 min,允许雄性小鼠在其包围物中探索大鼠。
      4. 5分钟后,将两只动物放回其家笼中。

  3. 小鼠的习惯化习惯
    这种模式模拟小鼠可能经历整个集落的短暂,频繁的相互作用(Dantzer等人,1987; Winslow和Camacho,1995)。 它包括五个短的探索会话,每个有一个分钟持续时间。
    1. 将主题鼠标放入与家庭相同的干净的塑料笼子里 笼中30分钟,然后测试以允许动物习惯   笼。
    2. 将一个新的幼年男性特异性进入笼子 与受试小鼠1分钟。 一分钟后取出刺激 鼠标,并将其放入保持笼
    3. 经过10分钟的延迟,   将相同的刺激小鼠放回与受试者的笼子 老鼠。 重复这两次,总共4个探索会话。
    4. 经过第四个十分钟的延误,第五次探索会话,将一个新颖的少年特异性放入笼子里 主题小鼠。 一分钟后,从笼子里删除刺激小鼠。

  4. 数据分析
    1. 使用秒表,记录主题鼠标的时间量 花费调查刺激动物。 刺激的调查 动物被定义为受试小鼠在一厘米内 将其头部指向另一只动物的受试小鼠, 接触或舔其他动物(图3,视频1)。 最近, Arakawa等人 (2014)也开发了一个自动化软件 用于检测社交互动模式,如果这种方法是 首选。 该软件显示产生与人类相似的结果 观察,但可能不适用于所有情况(Arakawa等人2014)。


      图3.社会识别任务的数据分析。 此图表说明了主题的调查标准 老鼠。对于这两个例子,受试小鼠必须在1cm内 刺激动物,其面部指向刺激。在他的 面板示出了研究的主题小鼠的实例 刺激小鼠的面部区域。 B.这是主题的图片 小鼠调查刺激小鼠的面部区域。 C.这 面板示出了研究的主题小鼠的实例 刺激小鼠的生殖器区域。 D.在这张图片中, 刺激小鼠正在调查刺激的肛门生殖区 鼠标。

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    2. 记录时间(以秒为单位)。
    3. 根据基因型,性别或治疗将动物分组。
    4. 平均勘探时间并确定标准误差。
    5. 使用标准误差测量值绘制数据作为误差条。
    6. 训练会话和召回会话之间的差异可以 使用Student's T检验确定。 据说动物有 形成了刺激动物的记忆,如果有显着的话 减少调查熟悉的刺激花费的时间   动物从训练阶段到召回阶段。 差异 可以通过ANOVA确定。

代表数据

  1. 社会认可任务
    图4是在社交识别范例期间收集的代表性数据的示例,其中年轻男性具有24小时延迟。图4A和4B是来自在回忆阶段中的新动物匹配的组的代表性数据;这是实验的对照组。以秒为单位记录探测时间,并且每个组一起平均,并计算标准误差。然后绘制该数据。预期受试动物花费相似的时间来研究训练阶段中的新动物以及回忆阶段中的新动物。这表明与熟悉的动物的探索时间的任何减少是由于熟悉的动物的记忆。图4C和4D是在召回阶段来自与相同小鼠配对的组的代表性数据。该实施例中的野生型小鼠在召回阶段显着减少了熟悉的小鼠的探索,表明刺激动物的记忆。这个显着的减少表明野生型小鼠形成了针对特异性的记忆。 NR2A小鼠已知具有受损的社会识别记忆,并且在24小时延迟后的回忆阶段中没有显示熟悉的小鼠的探索时间的显着减少(Jacobs和Tsien,2014)。通过学生T检验确定相之间的显着性,并通过ANOVA检验确定各组之间的差异。


    图4.男性特异性识别的代表性数据。 A。对于野生型和NR2A转基因小鼠的幼年雄性同时任务的代表性探索时间,以秒计。在两个会话中使用相同的幼年男性同种型。 B.用SEM误差棒图表示探测时间的平均值。 C.对于野生型和NR2A转基因小鼠的幼年雄性同时任务的代表性探索时间,以秒计。在召回会议中提出了一种新颖的少年男性特异性。 D.用SEM误差条图表示探测时间的平均值。在召回会议中提出了一个新颖的少年特异性
  2. 小鼠的习惯化习惯
    图5是在社会认知的习惯 - 习惯范例期间收集的代表性数据的示例。在该范例中,将受试小鼠引入一个特异性刺激小鼠四次,每次一分钟,随后用新型小鼠进行一次一分钟的探查。最后一次会议作为对前四届会议期间对熟悉刺激的探测预期减少的控制。图5A是表听所有五项试验的代表性数据。预期试验一到四的探索时间随着刺激特异性变得对受试小鼠熟悉而减少。将每组的每次探索的数据进行平均,并计算标准误差。图5B是通过试验数绘制的每组的平均勘探。连续试验之间的显着性通过Student's T检验确定,并且通过ANOVA检验确定各组之间的差异。


    图5.习惯 - 习惯范式的代表数据。 A。对于野生型和NR2A转基因小鼠的每个习惯 - 习惯范例的代表性探索时间,以秒为单位。在第四届会议中使用相同的幼年男性同种异体,在第五届会议中使用新颖的男性同种异体。 B.用SEM误差棒图表示探测时间的平均值。

笔记

尽管所描述的协议相对简单,并且涉及很少的专用设备,但是正确的执行和对细节的注意对于正确的结果和可重复性是必要的。

  1. 首先,实验设置应该在一个稍微暗淡,安静房间作为老鼠经常表现出对明亮的地区的恐惧,可以吃惊 通过突然的大声噪声或运动(Liu等人,2014)。
  2. 类似地,动物应习惯于空笼子和 空的房间,以避免动物表现出恐惧症(Deacon,2006)。   不要将相同的笼子与第一伴侣的粪便和尿液重复使用 可能存在于床上,给出不一致或不对称的结果 (Arakawa等人,2008; Tolokh等人,2013)。 由于使用大鼠笼 大鼠需要的丝网封壳的尺寸。 务必 使受试小鼠在训练前习惯于该测试笼 协议。
  3. 列车阶段和列车阶段之间的持续时间 调用相位根据所测试的存储器类型而变化。 通常对于短期记忆,持续时间为1小时或两小时 小时。 对于长期记忆,典型的持续时间为12-24小时
  4. 刺激动物选择是重要的几个原因。少年 雄性小鼠用于降低成年受试者的可能性 小鼠表现出攻击性行为(Hlinak和Krejci,1991; Heinla等人,2014)。小鼠品系的社会攻击性可能不同 [即:CD-1小鼠(Golden等人,2011),Tg2576小鼠,阿尔茨海默病 菌株(Alexander等人,2011),和具有改变的5-羟色胺水平的小鼠 (Takahashi等人,2012)],并且可能需要更多的侵袭性菌株 分离以避免战斗(Levine等人,1979; Serri和Ely,1984)。 此外,单独安置或已被用于的小鼠 育种可能更具侵略性。在勘探试验期间战斗 对于刺激小鼠和受试小鼠都是危险的。如果 战斗被观察到试验应立即停止 动物分开避免伤害。
  5. 社会认可可能 如果刺激小鼠在不同的菌株上则减少(Macbeth等人, ,2009; Jacobs和Tsien,2012; Jacobs和Tsien,2014)。女性 用作刺激动物应该在相同的遗传背景下由于观察到野生雌性小鼠更有可能繁殖 与来自相同遗传背景的雄性(Macbeth等人,2009)。
  6. 在这里描述的社会认可协议中,育种是 不发生。 电线外壳允许雄性鼠标探索 没有安装或交配行为的女性的气味。
  7. 在里面   使用大鼠来研究种间物种 承认。 可以使用幼年雄性大鼠,并且可能是优选的 尺寸较小。 大鼠包围防止大鼠和老鼠有   作为大鼠的直接相互作用和防止大鼠的杀死(Noack等人 al。,2010)。 确保彻底清洁动物之间的所有外壳 和与70%乙醇或适当的替代的会话。

致谢

这项工作由国家心理健康研究所(MH060236),国家老龄化研究所(AG024022,AG034663& AG025918),USAMRA00002和佐治亚研究联盟(全部给JZT)的资金支持。 该协议改编自(Jacobs和Tsien,2014; Jacobs等人,2015)。

参考文献

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How to cite this protocol: Jacobs, S. A., Huang, F., Tsien, J. Z. and Wei, W. (2016). Social Recognition Memory Test in Rodents. Bio-protocol 6(9): e1804. DOI: 10.21769/BioProtoc.1804; Full Text



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Stephanie A. Jacobs的其他实验方案(1)
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