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Apr 2019

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Assessing Rough-and-tumble Play Behavior in Juvenile Rats
幼年大鼠追逐打闹行为分析   

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Abstract

Play is a complex social behavior that is highly conserved across mammals. In most species, males engage in more frequent and vigorous play as juveniles than females, which reflects subtle yet impactful sex differences in brain circuitry and development. In this protocol, we describe a behavioral testing paradigm to assess social play in male and female juvenile rats. We highlight the behavior scoring criteria for distinguishing rough-and-tumble play from other play-related social behaviors. By analyzing both sexes, play behavior can be leveraged as a powerful tool to understand the sex-specific development and expression of social behavior.

Keywords: Social play (社会性游戏), Sex differences (性别差异), Behavior (行为), Brain development (大脑发育), Rats (大鼠), Social behavior (社会行为), Play fighting (打斗), Rough-and-tumble play (追逐打闹)

Background

Play is a highly conserved social behavior expressed by juveniles in several mammalian species, from rodents to humans (Auger and Olesen, 2009). Play is highly complex, involving dynamic interactions between conspecifics and is necessary for the development of appropriate social, cognitive, and affective behaviors later in life (Van den Berg et al., 1999; Von Frijtag et al., 2002; Baarendse et al., 2013). In many species, males engage in more frequent and vigorous play, called rough-and-tumble play or play fighting. This sex difference in behavior is developmentally programmed and is the result of sexual differentiation of the brain (Auger and Olesen, 2009; Thornton et al., 2009).

Here we describe a protocol to assess juvenile play using male and female rats. Each day throughout the juvenile period, rats are paired with an age-, sex-, and treatment-matched partner and play behavior is analyzed. In this way, this protocol is used to assess sex differences in baseline or “naturalistic” play. When combined with early-life pharmacological or genetic manipulation, this protocol can be used to study the processes of brain sexual differentiation and the development of social behavior circuitry (Vanderschuren et al., 2016; VanRyzin et al., 2019). Alternatively, the protocol can be performed as a single-trial to study sex differences in the activation of neural circuitry underlying play (Veenema et al., 2013; Bredewold et al., 2018).

Materials and Reagents

  1. Male and female juvenile (4-5-week-old) Sprague-Dawley rats
    Notes:
    1. We breed and maintain our own colony at the University of Maryland School of Medicine. This facilitates early-life treatments with pharmacological agents predicted to affect masculinization and avoids stress due to animal shipping or relocation. Animals are kept on a 12:12 h reverse light:dark cycle.
    2. Rats are tested as sex-, treatment- and age-matched non-sibling pairs. These “play pairs” will remain constant across multiple days of testing such that each animal is playing with the same play partner every day. To achieve this, we plan experiments to include rats from at least two separate litters. Depending on the number of experimental conditions, we will test at least two cohorts of play pairs (2 sets of paired litters, or 4 litters total) (see Figure 1A for experimental design and timeline).
    3. At birth, litter size is culled to 12-14 pups per dam to avoid confounds of over/under nutrition and maternal care. Litters are kept as sex- and treatment-balanced as possible, and litters with an initial sex ratio of > 2:1 males:females (or vice versa) are not used in our experiments due to potential in utero effects of prenatal testosterone.
  2. Pharmacological agents of interest
    Note: Depending on the hypothesis under study and the experimental timing of treatments, animals may need to be treated much earlier in life (prenatal exposure, postnatal masculinization, etc.; see VanRyzin et al., 2019 for examples).


    Figure 1. Representative experimental timeline and behavior illustrations. A. On the day of birth, pups from two litters are sexed, culled and treated (if necessary), and assigned to sex- and treatment-matched pairs with the opposite litter. The pups are allowed to grow undisturbed until the juvenile age. Beginning at P26, play behavior can be assessed daily throughout the juvenile period. B. Illustrations depicting rats engaging in the three main rough-and-tumble play behaviors–pouncing, pinning, and boxing.

Equipment

  1. Custom plexiglass testing arena 49 cm x 37 cm, 24 cm high (see Figure 2A)
  2. Behavioral testing room equipped with red light
  3. One to two lamp stands with red light bulbs for illumination of the testing arenas
  4. Video recording device and tripod stand
    Note: We typically use a smart phone with a tripod adapter to record behavioral tests.
  5. TEK-Fresh bedding (Envigo, catalog number: 7099)
  6. Fur-marking pen or permanent marker (Stoelting, catalog number: 50440-1)
  7. Stopwatch or timer
  8. Cleaning solution
    Note: Several different cleaning solutions may be used to sanitize the arenas after testing. We typically use MB-10 (Quip Laboratories).

Procedure

  1. Prior to the first test day, set up the behavioral testing room.
    1. Ensure that the red light illumination provides appropriate contrast for video recording.
    2. If testing more than one pair of animals at a time, arrange all testing arenas such that they can be adequately recorded by the available recording equipment. Using a smart phone camera, we are able to record 4 arenas simultaneously with sufficient detail and resolution. See Figure 2B.


      Figure 2. Testing arena and set up. A. Image of a single behavior testing arena. B. Image of four testing arenas arranged in front of a tripod for video recording.

    3. Fill each testing arena with enough TEK-fresh bedding to cover the bottom of the arena (approximately 2-3 cm deep).
    4. Plan play pairs and the order of behavioral testing. The order of testing needs to be changed each day to counterbalance the time of testing for each group across all test days.
  2. On the day of testing, move the animals (in their home cage) from the colony room into the behavioral testing room to allow them to habituate to the experimental environment (approx. 20 min prior to testing and at least 1 h after the start of the dark cycle). Testing during the dark phase (the active phase for the rat) is essential for proper behavioral analysis.
  3. Mark one of the animals in each play pair either with a fur-marking pen (on the head or back) or by striping the tail with a permanent marker. This facilitates behavioral analysis as it is easier to attribute specific behaviors to the marked or unmarked animal.
  4. Once the animals have habituated, place an animal pair into the testing arena and allow the animals to acclimate to the arena for 2 min.
  5. After acclimation, record behavior for 10 min.
  6. Once the test has concluded, stop the recording. Remove the animals from the testing arena and place them back in their home cage.
  7. Repeat Steps 4-6 as many times as needed until all animals have been tested.
  8. On subsequent days, repeat Steps 2-7. We typically perform the test for 4 continuous days (P27-30) to capture the peak sex difference in play behavior; however, the test can be repeated each day throughout the entire juvenile period (approximately P26-35).
Notes:
  1. During the test, the experimenter should remain behind a curtain or out of sight of the testing arenas. Additionally, maintain quiet in the room as external noises may disrupt behavior.
  2. It is not necessary to clean the testing arena or change the bedding between trials. At the conclusion of each test day, the arena should be cleaned (using a cleaner of the experimenter’s choosing) and bedding replaced in preparation for the next day.
  3. Be sure to keep the red light intensity to a minimum to avoid creating an artificially stressful testing environment. The lighting for our behavior testing is typically < 50 lux. Additionally, red light should be used for Sprague Dawley rats as they are albino and their inability to see red light keeps the testing environment as close to a natural dark phase as possible for optimal behavior testing.
  4. While rats do emit ultrasonic vocalizations during play, this does not impact the expression of play (Kisko et al., 2015), and does not confound testing more than 1 pair of animals at a time.

Data analysis

  1. Behavior is scored offline by an experimenter blind to the sex and treatment of the animals.
    Note: Scoring may be done by hand or by using keylogger software.
  2. The animal performing a given behavior receives a score for that behavior. Thus, one play pair yields two data points. This helps improve interpretability of the data, as high- and low-playing animals will not be averaged with their play partner’s behavior. Moreover, scoring behavior for each animal allows for an analysis of each individual animal over time to get an average play value across all test days.
  3. To assess rough-and-tumble play behavior, we score the following behaviors (see Videos 1-3 and Figure 1B):
    1. Pouncing–Scored when one animal launches itself toward the other’s nape or back. This is often a vigorous motion and is scored to the animal performing the pounce.
      Note: Animals may forcefully bump into each other or perform “low effort” pounces. These are generally not scored as the intent to initiate play is much more ambiguous.
    2. Pinning–Scored when a pouncing animal flips the other completely onto its back (all four limbs off the ground). A pin is scored to the pouncing animal, and often will be scored directly after a pounce.
      Note: Pinning is often the result of the animal that is being pounced on continuing the play bout by rolling onto their back in a defensive position. However, the animal may flip over onto its side, keeping weight on their feet. In these cases, a pin is not scored.
    3. Boxing–Scored when both animals rear up on their hind legs and paw at each other. Both animals receive a score for boxing.
      Note: Boxing is much less frequent than either pouncing or pinning and is observed often following bouts of chasing, when the animal being chased turns and confronts the chaser.

      Video 1. Sample behavioral sequence featuring pouncing and pinning. (All animal procedures described here are performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine (Protocol # 0517007, 06/30/20).)

      Video 2. Sample behavioral sequence featuring boxing. (All animal procedures described here are performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine (Protocol # 0517007, 06/30/20).)

      Video 3. Sample video of an entire play behavior test. (All animal procedures described here are performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine (Protocol # 0517007, 06/30/20).)

  4. The following behaviors can also be scored but are considered to be social or locomotor play behaviors:
    1. Chasing Scored when one animal actively chases the other. Only the “chaser” receives a score for chasing.
      Note: Typically, chasing precedes pounces or boxing behavior.
    2. Bouncy gait–Scored when one animal moves around the arena in a series of “jumps” or “hops”.
  5. The following non-play social behaviors can also be scored:
    1. Anogenital sniffing–Scored when one animal actively sniffs the anogenital region of the other.
    2. Licking and grooming–Scored when one animal actively licks or grooms the head or anogenital region of the other.
    3. Huddling–Scored when both animals are sitting in very close proximity/touching each other but are not engaged in any other form of social behavior (sniffing/licking/grooming). Both animals receive a score for huddling.
    4. Crawling over/under–Scored when one animal crawls over or under the other.
      Note: This typically happens during periods of environmental exploration.
  6. The following environmental exploration behaviors can also be scored:
    Note: These behaviors are best scored as a duration of time the animal spends engaged in each category.
    1. Digging and burrowing in the bedding–Scored as the duration of time one animal burrows or nests in the bedding.
    2. Chewing on the bedding–Scored as the duration of time one animal holds a piece of bedding in their paws and chews on it.
    3. General arena investigation–Scored as the duration of time one animal spends sniffing or rearing up on their hind legs to explore the walls of the testing arena.
  7. Statistical Analysis:
    1. If age is an experimental factor of interest, the data may be represented as an average for each day and analyzed using a repeated-measures ANOVA (see Figure 3A and Argue and McCarthy, 2015 for more examples).
    2. If age is not of interest, the data may be averaged across all days for an individual animal and analyzed using a t-test or ANOVA depending on the number of experimental groups (see Figures 3B-3E and VanRyzin et al., 2019 for more examples).


      Figure 3. Sample data. A. Sample data showing total play (pounces, pins, and boxes) for males and females daily from P27-P30. B. Sample data showing the total play data from A as an average across all four testing days. C-E. Sample data from B broken down into pounces (C), pins (D), and boxes (E). Data are represented as the mean ± SEM. Blue and red lines/bars indicate males and females, respectively. Open circles indicate values for individual animals. * = P < 0.05, ** = P < 0.01.

Notes

  1. Prior to the first test day, animals can be tested in an open field test for general locomotor activity and exploratory behavior to rule out any confounds of activity differences between groups. In general, we have never found baseline activity differences between males and females, but experimental manipulations may affect this.
  2. Filling the testing arena with too much bedding can lead to a decrease in social behavior and an increase in environmental exploration, as the animals will begin to burrow under the bedding.
  3. We test animals on a reverse light:dark cycle and begin behavioral testing 1-2 h after the onset of the dark cycle. It is important to keep the timing of behavioral analysis consistent across days to avoid any circadian influences on behavior.
  4. Depending on the number of test days, age of the animals, and pharmacological manipulations, animals may begin to display sex behavior at the onset of puberty (approximately P40 in our colony). We do not score sex behavior as a social behavior in this paradigm, as it occurs very rarely in our experience; however, if it is occurring frequently, it can be analyzed as a separate set of behaviors to determine if puberty has occurred early.
  5. Male and female play behavior is expressed along a continuum from high to low play. While males, on average, are on the “high” end of the continuum relative to females, there is a fair amount of natural individual variability. Each cohort of animals will likely have a few high-playing females and low-playing males. This natural behavioral variability is not a reason to exclude these animals from analysis; rather, plan experiments with a sufficient sample size to account for this. We recommend at least 8 animals per sex.
  6. Play pairs should be same-sex, same-treatment. Mixing sexes or treatments within a given play pair may result in an intermediate play phenotype (i.e., a more playful partner will increase a low playing rats play and vice versa). See Argue and McCarthy, 2015 for an example.

Acknowledgments

This work was funded by the National Institutes of Health R01MH52716 and R01DA039062 to M.M.M.This protocol was adapted from Argue and McCarthy, 2015, and Vanryzin et al., 2019.

Competing interests

The authors have no competing interests to declare.

Ethics

All animal procedures described here are performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine (Protocol # 0517007, 06/30/20).

References

  1. Argue, K. J. and McCarthy, M. M. (2015). Characterization of juvenile play in rats: importance of sex of self and sex of partner. Biol Sex Differ 6: 16.
  2. Auger, A. P. and Olesen, K. M. (2009). Brain sex differences and the organisation of juvenile social play behaviour. J Neuroendocrinol 21(6): 519-525.
  3. Baarendse, P. J. J., Counotte, D. S., O’Donnell, P. and Vanderschuren, L. J. M. J. (2013). Early social experience, is critical for the development of cognitive control and dopamine modulation of prefrontal cortex function. Neuropsychopharmacol 38:1485-1494.
  4. Bredewold, R., Nascimento, N. F., Ro, G. S., Cieslewski, S. E., Reppucci, C. J., and Veenema, A. H. (2018). Involvement of dopamine, but not norepinephrine, in the sex-specific regulation of juvenile socially rewarding behavior by vasopressin. Neuropsychopharmacol 43:2109-2117.
  5. Kisko, T. M., Euston, D. R. and Pellis SM. (2015). Are 50-khz calls used as play signals in the playful interactions of rats? III. The effects of devocalization on play with unfamiliar partners as juveniles and as adults. Behav Processes 113:113-121.
  6. Thornton, J., Zehr, J. L. and Loose, M. D. (2009). Effects of prenatal androgens on rhesus monkeys: a model system to explore the organizational hypothesis in primates. Horm Behav 55(5): 633-645.
  7. Vanderschuren, L. J., Achterberg, E. J. and Trezza, V. (2016). The neurobiology of social play and its rewarding value in rats. Neurosci Biobehav Rev 70: 86-105.
  8. Van den Berg, C. L., Hol, T., Van Ree, J. M., Spruijt, B. M., Everts, H. and Koolhas, J. M. (1999). Play is indispensable for an adequate development of coping with social challenges in the rat. Dev Psychobiol 34(2):129-138.
  9. Vanryzin, J., Marquardt, A., Argue, K., Vecchiarelli, H. A., Ashton, S., Arambula, S. E., Hill, M. N. and McCarthy, M. M. (2019). Microglial phagocytosis of newborn cells is induced by endocannabinoids and sculpts sex differences in juvenile rat social play. Neuron 102(2):435-449.
  10. Veenema, A. H., Bredewold, R. and De Vries, G. J. (2013). Sex-specific modulation of juvenile social play by vasopressin. Psychoneuroendocrinology 38(11): 2554-2561.
  11. Von Frijtag, J. C., Schot, M., van den Bos, R. and Spruijt, B. M. (2002). Individual housing during the play period results in changed responses to and consequences of a psychosocial stress situation in rats. Dev Psychobiol 41(1): 58-69.

简介

游戏是一种复杂的社会行为,在哺乳动物中高度保守。在大多数物种中,雄性幼鱼比雌性更频繁,更有活力,这反映出大脑回路和发育中的细微但有影响的性别差异。在此协议中,我们描述了一种行为测试范式来评估雄性和雌性幼鼠的社交游戏。我们重点介绍了行为评分标准,以区分粗鲁的游戏和其他与游戏相关的社交行为。通过分析性别,可以将玩耍行为作为一种有力的工具来理解针对性别的发展和社会行为的表达。
【背景】游戏是青少年在从啮齿动物到人类的几种哺乳动物中表达的高度保守的社会行为(Auger and Olesen,2009)。玩耍非常复杂,涉及特定对象之间的动态互动,是以后生活中适当的社交,认知和情感行为发展所必需的(Van den Berg et al。,1999; Von Frijtag 等人。,2002; Baarendse等人,,2013)。在许多物种中,雄性参与更频繁,更剧烈的游戏,称为“粗暴打法”或“打斗”。这种行为上的性别差异是发育程序性的,是大脑性别分化的结果(Auger和Olesen,2009; Thornton et al。,2009)。

在这里,我们描述了一种使用雄性和雌性大鼠评估青少年游戏的协议。在整个少年时期的每一天,都将大鼠与年龄,性别和治疗匹配的伴侣配对,并分析游戏行为。通过这种方式,该协议可用于评估基线或“自然”游戏中的性别差异。当与早期药理或基因操作相结合时,该协议可用于研究大脑性别分化的过程和社会行为电路的发展(Vanderschuren et al。,2016; VanRyzin 等,2019)。另外,该协议也可以作为单项试验执行,以研究游戏中潜在的神经回路激活中的性别差异(Veenema等,2013年; Bredewold等(等)。 ,2018)。

关键字:社会性游戏, 性别差异, 行为, 大脑发育, 大鼠, 社会行为, 打斗, 追逐打闹

材料和试剂

  1. 雄性和雌性少年(4-5周龄)Sprague-Dawley大鼠
    注意:
    1. 我们在马里兰大学医学院繁殖并维持自己的殖民地。这有助于使用预计会影响男性化的药理剂进行早期治疗,并避免了由于动物运输或搬迁而产生的压力。将动物置于12:12 h的逆光:黑暗周期中。
    2. 测试鼠是按性别,治疗和年龄相匹配的同胞对进行测试。这些“游戏对”在测试的多天中将保持不变,以使每只动物每天都与同一个游戏伙伴一起玩。为了实现这一目标,我们计划进行实验,以包括至少两个单独的猫砂中的大鼠。根据实验条件的数量,我们将测试至少两个游戏对(2组成对的垫料,或总共4个垫料)(有关实验设计和时间表,请参见图1A)。
    3. 出生时,每只水坝的产仔数被淘汰至12-14头,以避免营养和孕产妇护理过度/不足的混杂。垫料应保持性别和治疗方面的平衡,垫料的初始性别比应大于或等于0。我们的实验未使用2:1的男性:女性(或相反),因为产前睾丸激素可能会在子宫内产生作用。
  2. 感兴趣的药理剂
    注意:根据研究假设和治疗的实验时间,可能需要在生命中更早地对动物进行治疗(产前暴露,产后男性化等;请参见VanRyzin 等)。 ,例如2019)。


    图1.代表性的实验时间表和行为说明。 A.出生当天,对两窝幼崽进行性别,剔除和处理(如有必要),并分配给与性别和治疗匹配的配对与相反的垃圾。允许幼犬不受干扰地生长直至幼年。从P26开始,可以在整个少年时期每天评估比赛行为。B.插图描绘了老鼠参与三种主要的粗糙和摔打行为-突击,钉扎和拳击。

设备

  1. 定制有机玻璃测试场49 x 37厘米,高24厘米(见图2A)
  2. 行为测试室配备红灯
  3. 一到两个带红色灯泡的灯架,用于照亮测试场所
  4. 录像设备和三脚架支架
    注意:我们通常使用带三脚架适配器的智能手机来记录行为测试。
  5. TEK-新鲜床上用品(Envigo,货号:7099)
  6. 毛笔或永久性记号笔(Stoelting,目录号:50440-1)
  7. 秒表或计时器
  8. 清洁液
    注意:测试后,可以使用几种不同的清洁溶液对竞技场进行消毒。我们通常使用MB-10(Quip实验室)。

程序

  1. 在第一个测试日之前,请建立行为测试室。
    1. 确保红光照明为视频录制提供适当的对比度。
    2. 如果一次要测试一对以上的动物,请安排所有测试场所,以便可以使用可用的记录设备充分记录它们。使用智能手机摄像头,我们可以同时以足够的细节和分辨率记录4个舞台。参见图2B。


      图2.测试场所和设置。 A.单个行为测试场所的图像。B.放置在三脚架前的四个测试场地的图像进行录像。

    3. 在每个测试区域中填充足够的TEK新鲜寝具,以覆盖区域底部(大约2-3厘米深)。
    4. 计划游戏对和行为测试的顺序。每天都需要更改测试顺序,以平衡所有测试日中每个组的测试时间。
  2. 在试验当天,将动物(放在笼中)从殖民地房间移入行为试验室,以使其适应实验环境(试验前约20分钟,开始试验后至少1小时)。黑暗周期)。在黑暗阶段(大鼠的活跃阶段)进行测试对于正确的行为分析至关重要。
  3. 用毛皮标记笔(在头上或后面)或用永久性标记划去尾巴,标记每对游戏中的一只动物。这有助于进行行为分析,因为更容易将特定行为归因于标记或未标记的动物。
  4. 一旦动物适应了环境,就将一对动物放入测试场地,让它们适应场地2分钟。
  5. 适应后,记录行为10分钟。
  6. 测试结束后,停止记录。从测试场所中取出动物,然后将它们放回他们的家笼中。
  7. 根据需要重复步骤4-6多次,直到所有动物都经过测试。
  8. 在随后的几天,重复步骤2-7。我们通常会连续4天(P27-30)进行测试,以捕获游戏行为中的性别差异峰值;但是,可以在整个青少年时期(大约P26-35)每天重复进行该测试。
注意:
  1. 在测试过程中,实验人员应保持在窗帘后面或视线范围之外。此外,请保持房间安静,因为外部噪音可能会干扰行为。
  2. 不必在测试之间清洁测试场地或更换被褥。在每个测试日结束时,应清洁赛场(使用实验人员选择的清洁剂),并更换被褥,为第二天的比赛做准备。
  3. 确保将红光强度保持在最低水平,以避免造成人为的压力测试环境。用于我们的行为测试的照明通常&lt; 50勒克斯 此外,Sprague Dawley大鼠由于患有白化病,因此应使用红光,并且由于无法看到红光而使测试环境尽可能接近自然暗相,以进行最佳行为测试。
  4. 尽管大鼠在游戏过程中会发出超声波发声,但这不会影响游戏的表达(Kisko 等人 ,2015),并且不会混淆测试一次有1对动物。

数据分析

  1. 行为是由不了解动物性别和治疗方法的实验者离线评估的。
    注意:评分可以手动完成,也可以使用键盘记录器软件完成。
  2. 进行给定行为的动物会获得该行为的分数。因此,一个游戏对产生两个数据点。这将有助于提高数据的可解释性,因为高低玩耍的动物不会根据其玩伴的行为进行平均。此外,对每只动物的评分行为可以对每只动物进行一段时间的分析,从而获得所有测试日的平均游戏价值。
  3. 为了评估粗暴的比赛行为,我们对以下行为进行了评分(请参见视频1-3和图1B):
    1. 突袭-当一只动物朝另一只动物的颈背或后退时得分。这通常是一种剧烈的动作,会被执行突袭动作的动物所打分。
      注意:动物可能会强行撞向对方或进行“低强度”突袭。由于开始比赛的意图更加含糊不清,因此通常不予评分。
    2. 固定-当有突袭动物将另一只动物完全向后翻转时(所有四肢都离开地面)时得分。会为刺中的动物划上一个别针,通常在突刺后会直接被刻上。
      注意:钉扎通常是由于动物在防守端继续向后滚动而被猛扑而导致的。但是,动物可能会翻身,使其脚保持重量。在这种情况下,不会得分。
    3. 拳击-当两只动物的后腿都站起来并互相掌着时,得分。两只动物的拳击得分都很高。
      注意:与追逐或钉扎相比,拳击的频率要低得多,当追逐的动物转弯并面对追逐者时,经常会在追逐追击时观察到。


      视频1.具有突击和固定功能的示例行为序列


      视频2.具有拳击功能的示例行为序列


  4. 以下行为也可以评分,但被认为是社交或自发游戏行为:
    1. 追逐当一只动物主动追逐另一只时得分。只有“追逐者”获得追逐分数。
      注意:通常,追逐先于突袭或拳击行为。
    2. 有弹性的步态-当一只动物以一系列“跳跃”或“跳跃”的形式在赛场上移动时得分。
  5. 还可以对以下非游戏性社交行为进行评分:
    1. 肛门生殖器嗅探-当一只动物主动嗅探另一只动物的肛门生殖器区域时评分。
    2. 舔和修饰–当一只动物积极舔或修饰另一只动物的头部或生殖器部位时得分。
    3. 挤在一起-当两只动物都非常靠近/彼此接触却没有参与任何其他形式的社交行为(嗅/舔/修饰)时得分。两只动物都缩得分。
    4. 爬上/爬下-当一只动物爬上另一只或下时得分。
      注意:这通常发生在环境探索期间。
  6. 还可以对以下环境探索行为进行评分:
    注意:这些行为最好按照动物花在每个类别上的时间进行评分。
    1. 在床上挖掘和挖洞–记为一只动物在床上挖洞或筑巢的持续时间。
    2. 咀嚼床上用品-记为一只动物在其爪中握住一块床上用品并对其进行咀嚼的持续时间。
    3. 常规竞技场调查-评分为一只动物嗅探或抬起后腿探究测试竞技场墙壁的持续时间。
  7. 统计分析:
    1. 如果年龄是感兴趣的实验因素,则数据可以表示为每天的平均值,并使用重复测量方差分析进行分析(更多示例,请参见图3A和Argue和McCarthy,2015年)。
    2. 如果不关心年龄,则可以根据实验组的数量,对单个动物的整天数据进行平均,并使用 t 检验或ANOVA进行分析(请参见图3B-3E和VanRyzin 等,2019年获得更多示例)。


      图3.样本数据。 A.样本数据显示了P27-P30每天男性和女性的总运动量(突跳,大头钉和长方体)。B.样本数据显示了所有四个测试日中A的总比赛数据的平均值。CE。来自B的样本数据分为突点(C),大头针(D)和方框(E)。数据表示为平均值±SEM。蓝色和红色线条/条分别表示男性和女性。空心圆圈表示单个动物的值。* = P &lt; 0.05,** = P &lt; 0.01。

笔记

  1. 在第一个测试日之前,可以在野外测试中对动物进行一般运动活动和探索行为的测试,以排除各组之间活动差异的任何混淆。通常,我们从未发现男性和女性之间的基线活动差异,但是实验操作可能会影响这一点。
  2. 由于动物将开始在被褥下挖洞,因此在试验场上铺满过多的被褥会导致社交行为的减少和环境探索的增加。
  3. 我们在逆光:黑暗周期中测试动物,并在黑暗周期开始后1-2小时开始进行行为测试。重要的是要保持行为分析的时间跨几天一致,以避免任何昼夜节律对行为的影响。
  4. 根据测试天数,动物的年龄和药理操作,动物可能会在青春期开始时开始表现出性行为(在我们的殖民地中约为P40)。在这种范例中,我们不将性行为视为一种社会行为,因为在我们的经验中这种情况很少发生。但是,如果它频繁发生,则可以将其作为一组单独的行为进行分析,以确定青春期是否较早发生。
  5. 男性和女性的游戏行为沿从高到低的连续性表达。平均而言,男性相对于女性处于连续体的“高端”,但自然个体变异性却相当大。每组动物可能都会有一些高玩性雌性和低玩性雄性。这种自然的行为变异性并不是将这些动物排除在分析范围之外的原因。而是计划具有足够样本量的实验来说明这一点。我们建议每性至少8只动物。
  6. 游戏对应该是同性,同等待遇。在给定的游戏对中混合性别或待遇可能会导致中间的游戏表型(即,更嬉戏的伴侣会增加低打的大鼠游戏,反之亦然)。有关示例,请参见Argue和McCarthy,2015年。

致谢

这项工作是由美国国立卫生研究院R01MH52716和R01DA039062资助MMM的,该协议改编自Argue和McCarthy,2015年以及Vanryzin等,2019年。

利益争夺

作者没有竞争利益要声明。

伦理

此处描述的所有动物操作步骤均应根据马里兰大学医学院动物护理和使用委员会的规定执行(协议编号0517007,06 / 30/20)。

参考文献

  1. Argue,KJ和McCarthy,MM(2015)。大鼠青少年游戏的特征:自我性别和伴侣性别的重要性。 生物性别差异 6:16。
  2. Auger,AP和Olesen,KM(2009)。脑性别差异和青少年社交游戏行为的组织。 J Neuroendocrinol 21(6):519-525。
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  4. R.Bredewold,Nascimento,NF,Ro,GS,Cieslewski,SE,Reppucci,CJ和Veenema,AH(2018)。多巴胺(而非去甲肾上腺素)参与针对青少年的社会奖励行为的性别特定调节 Neuropsychopharmacol 43:2109-2117。
  5. Kisko,TM,Euston,DR和Pellis SM。(2015)。 50-khz呼叫是否在大鼠的嬉戏互动中用作游戏信号?三,失恋对少年和成年后与陌生伴侣玩耍的影响。 行为过程 113:113-121。
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  7. Vanderschuren,LJ,Achterberg,EJ和Trezza,V.(2016)。社交游戏的神经生物学及其在大鼠中的有益价值。 Neurosci Biobehav启示录70:86-105。
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引用:VanRyzin, J. W., Marquardt, A. E. and McCarthy, M. M. (2020). Assessing Rough-and-tumble Play Behavior in Juvenile Rats. Bio-protocol 10(1): e3481. DOI: 10.21769/BioProtoc.3481.
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