参见作者原研究论文

本实验方案简略版
Feb 2020

本文章节


 

Induction of Repeated Social Defeat Stress in Rats
大鼠持续性社交失败压力诱导   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Repeated social defeat stress (RSDS) is a model of chronic stress in rodents. There are several variants of social defeat procedures that exert robust effects in mice, but few published detailed protocols to produce a robust stress and altered immunological profile in rats. In this article, we describe the protocol for the induction of RSDS in adult male Sprague-Dawley rats. Using a resident-intruder paradigm, a physical component of stress is induced by direct attack from the resident aggressive retired breeder Long-Evans rats on the intruder experimental rats. A subsequent threat component is induced by the presence of the aggressor in the vicinity of the intruder, but with physical separation between them. The RSDS induced by this protocol produces robust immunological and behavioral changes in the experimental rats, as evidenced by development of anxiety-like behaviors in open field, social interaction, and elevated plus maze tests, as well as by changes in immune parameters (Munshi et al., 2020). This approach has been used as an ethologically relevant model of stressors that are potent enough to impact neural circuits that are similar to the neural circuits impacted in patients with depression and anxiety.


Keywords: Aggressor rats (侵略鼠), Home cage (家笼), Intruder rats (侵略鼠), Psychological stress (心理压力), Social defeat (社交失败), Submission (提交)

Background

Repeated social defeat stress (RSDS) is a robust model of chronic psychological stress in rodents (Rygula et al., 2005; Berton et al., 2006; Liu et al., 2017). It causes robust depression-like behavioral changes including anxiety, anhedonia, and social-avoidance (Rygula et al., 2005 and 2006; Berton et al., 2006). A unique aspect of social defeat stress that distinguishes it from other environmental stressors is that the subjects do not develop habituation of pituitary-adrenal axis activity over repeated social confrontations (Tornatzky and Miczek, 1993; Koolhaas et al., 1997), unlike many other repeated stressors, even though the subjects generate behavioral stress responses (Tidey and Miczek, 1997).


Additionally, RSDS is known to have a robust effect on immune parameters that affect anxiety behavior in rodents (Wohleb et al., 2013). Furthermore, RSDS induces anxiety-like behavior by acting via interleukin-1 type 1 receptor (IL-1R1) in the endothelial cells in the brain (Wohleb et al., 2011). RSDS-induced macrophage trafficking in the brain is required for the development of anxiety behavior, which in turn is dependent on microglia activation and recruitment of primed monocytes to the brain (Wohleb et al., 2013). Myeloid cells isolated from mice undergoing RSDS have enhanced production of interleukins (IL) and other cytokines, such as IL-1β, TNF-α, and IL-6, following toll-like receptor stimulation with the immunogen lipopolysaccharide (Stark et al., 2002; Bailey et al., 2009; Powell et al., 2009).


Social defeat procedures have been leveraged to understand the neurobiology of anxiety or depressive behaviors by examination of its effects on limbic regions. RSDS has also been shown to decrease the firing of dopaminergic neurons projecting from the ventral tegmental area (VTA) to the medial prefrontal cortex (mPFC) in mice (Chaudhury et al., 2013). In addition, this kind of stress is also known to cause a persistent neuronal adaptation in the basolateral amygdala (BLA), hippocampus, and PFC; for example, RSDS caused a decrease in apical dendritic spine density in CA1 hippocampal neurons but not in BLA neurons in rats; it also caused dendritic atrophy of CA1 basal dendrites, while increasing dendritic arborization in BLA pyramidal neurons (Patel et al., 2018). A similar stress paradigm, called chronic social defeat stress (CSDS), has been shown to decrease spine density from apical dendrites of PFC pyramidal neurons but increase BLA dendritic arborization after one month (long-term); however, it also increased BLA stellate neuronal spike density in the short-term (Colyn et al., 2019).


There is significant evidence that some variations of social defeat can preferentially produce effects on appetitive motivated behaviors (described as rodent models of anhedonia/depressive behaviors; Riga et al., 2015; Yoshida et al., 2021) versus effects on open field and maze exploration (described as rodent models of anxiety). In this context, social defeat has shown a degree of predictive validity for antidepressant drugs (Tsankova et al., 2006; Golden et al., 2011). Similar social defeat stress paradigms have recently been studied in adolescent transgenic mice to explore genotype by environment interactions resulting in the development of phenotypes in psychiatric diseases in the “two-hit” model. For example, the mice overexpressing Tcf4 developed cognitive impairments and novelty-induced hyperactivity when exposed to social defeat stress (Volkmann et al., 2021). Social defeat stress has also been used in mice to test the pharmacological effects of psychotropic drugs to investigate stress-induced immune dysregulation. For example, one study has shown that the anti-asthenic drug bromantane (Ladasten) decreased depressive-like behavior by preventing development of avoidance behavior and also by improving locomotor activity after stress. Similarly, Ladasten also prevented the stress-induced shift of CD4/CD8 T-cells towards T-cytotoxic cells and normalized their ratio in the thymus, spleen and blood of mice (Tallerova et al., 2014).


While there are published protocols for social defeat in mice, social defeat in rats, particularly repeated social defeat, has its own set of challenges. There are few detailed protocols for a repeated social defeat procedure in rats that can reliably produce a stress phenotype and altered peripheral immune function. One purpose of this article is to fill this need. The RSDS model leverages ongoing social dynamics, instead of imposing circumstances that are unusual, which provides a degree of face validity, and can potentially provide more translatable information than less naturalistic stressors for understanding several of the physiological and behavioral abnormalities induced by social stress between individuals. In our original study (Munshi et al., 2020), we used the RSDS model in adult male Sprague-Dawley rats, following a previously published protocol (Jaisinghani and Rosenkranz, 2015). This has been adapted and modified from an earlier published study using the social defeat model of stress (Berton et al., 2006).


Subjects and housing: Adult male Sprague-Dawley rats (Envigo, Indianapolis, IN) were obtained at post-natal day 59-63 and were housed 2-3 per cage in a climate-controlled facility at Rosalind Franklin University of Medicine and Science, with ad libitum access to standard rat chow diet and water. After habituating in the animal facility for at least four days, rats were subjected to RSDS or control handling. The body-weights of the Sprague-Dawley rats were 300-350 g at the start of the experiments. Lights in the housing room were on a reverse 12 h light/dark schedule (lights off: 07:00-19:00). Sprague-Dawley rats in typical laboratory conditions are fairly social and rarely attack other rats, outside of play fighting or dams that are still lactating. Therefore, an alternative is required to induce defeat. Adult male retired breeder Long-Evans rats (Envigo, Indianapolis, IN) were single-housed and used to induce social defeat stress in the Sprague-Dawley rats. Rats were randomly assigned to experimental groups, and experiments were performed in multiple cohorts. The social defeat experiments were performed in a separate procedure room within the animal facility that was dedicated for stress procedures. This separate room was approximately 8 meters away from the housing room. The experiments were performed during the dark-phase of the rats’ diurnal cycle, between 9 am and 2 pm. Residents and intruders were not housed in the same cages, as is done for some social defeat procedures in mice. In our procedure, residents and intruders only interacted during the single daily stress sessions.


Because these experiments involve stress, it was particularly important to establish humane exclusion criteria that go beyond standard humane endpoints in consultation with our institutional veterinarian. Rats were monitored daily. This included daily weighing of the rats, observation of behavior (lethargy, extreme avoidance of the experimenter), and observation of appearance (coat appearance, presence of porphyrin). If any rat displayed a combination of >15% reduction in weight, persistent porphyrin staining, lethargy, or indication of non-grooming (matted fur), it would be removed from the study. Long-Evans rats were also monitored over the course of the social defeat procedures for the same measures (observation of behavior and of appearance).

Materials and Reagents

  1. Clean paper towels

  2. Intruder (experimental) adult male Sprague-Dawley rats

  3. Resident (aggressor) adult male retired breeder Long-Evans rats

  4. Scoring sheet (Table 1)

  5. 70% ethanol

  6. Disinfectant wipes for equipment (for example, Caviwipes Surface Disinfectant Wipes, Metrex item# 13-1100)


    Table 1. Scoring sheet.
    This is a standard scoring sheet that can be useful for the social defeat procedure. After submission, or if any criteria (a-c) are met, the Phase 1 session ends and the intruder rat is placed in the wire mesh enclosure. The Phase 2 session (separated by wire mesh enclosure) ends after 15 minutes. See completed sheet below for more details (Table 2).
    Resident Intruder Stress
    Protocol:
    Your Name:
    Resident rat + cage is placed in procedure room
    After at least 5 min, the Intruder rat is brought into the procedure room in a transport cage; the rat is placed in the cage with the Resident. Timer is started.
    After submission, the Intruder is placed into a smaller wire mesh enclosure within the Resident cage for 15 min
    END Phase 1 session after submission or:
    a. 15 min with no submission
    b. 10 attacks with no submission
    c. 5 min with no attacks
    END Phase 2 after:
    a. 15 min after placement in wire mesh enclosure
    Intruder (Stressed) Rat Number
    Date
    Resident Rat Number
    Start Time
    Time to First Attack
    Number of Attacks
    Submitted? (Y or N)
    Time Separated

































































































































    Comments:

Equipment

  1. Resident rat housing cages: sanitized woodchip bedding, 21” × 11.5” × 8” height, clear polycarbonate, wire top

  2. Rat transport cages: sanitized woodchip bedding, 12” × 6.5” × 5” height, clear polycarbonate, microisolator top

  3. Smaller wire mesh enclosure (Figure 3): approximately 6” × 7” × 8” height, 3/4” square mesh, PVC coated

  4. Digital stopwatch with time of day

Procedure

  1. RSDS by the resident-intruder paradigm is a robust model of chronic psychological stress in rodents (Rygula et al., 2005; Berton et al., 2006; Liu et al., 2017). In our study, housing cages of rats were randomly assigned to the stressed and control groups.

    Note: Most Long-Evans retired breeders are aggressive in our conditions, but some are not. You can screen these rats prior to experiments, using the same resident-intruder approach described below, to select more or less aggressive rats depending on your needs. If more aggressive resident behavior is required, pre-screening criteria may include short latency to attack and multiple attacks within the first minute of the session. In our experiments, the retired breeders displayed reduced attacks over the course of weeks-months. If a retired breeder displayed no attacks, it might be removed from the rotation of aggressive residents.

  2. The housing cages of the resident aggressor Long-Evans rats, containing one Long-Evans rat per cage, were transported to a dimly lit procedure room free from noise within the animal facility on the morning of the experiments. They were left undisturbed for acclimation in the environment for 15-30 min.

    Note: In our experience, a single experimenter can perform this procedure with up to 3 resident-intruder interactions at the same time (i.e., 3 resident cages in the procedure room). More than this becomes difficult to observe by one individual.

  3. Experimental Sprague-Dawley rats were weighed daily in their housing room and their body weight was noted. Animal condition was also noted, with attention to porphyrin around the eyes, nose, or fur.

  4. After weighing, the Sprague-Dawley rats were placed in clean transport cages.

    1. Control rats (housed together) remained in transport cages for 30 min, then they were returned to their home cage.

    2. Rats in the stress group were transported to the procedure room in clean transport cages separate from the controls.

  5. PHASE 1 (Direct physical contact): Individual Sprague-Dawley rats were transferred to the housing cage of a resident Long-Evans rat in the procedure room and the lid of the cage was secured tightly. This allowed the intruder rat to stay in direct physical contact with the resident rat (Figure 1). The time of this event was noted. The rats were continuously observed for the entire duration of the interaction.

    All scoring of a cohort of rats was performed by one trained experimenter. The experimenter was positioned at a distance that allows clear observation of the rats and rapid intervention, but far enough that rats do not direct attention towards the experimenter (approximately 1-2 meters). The measures can be reliably obtained by one experimenter, and we therefore did not routinely video sessions.



    Figure 1. Setting up the resident intruder procedure.

    A. Photographs of the resident cage with the wire mesh enclosure (green) placed inside (left). Also shown with resident present (right) for scale. B. Phase 1 of the stress procedure, when resident and intruder can have direct physical contact. C. Phase 2 of the stress procedure, when resident and intruder are separated by the wire mesh enclosure.


  6. During the period of direct physical contact, every attack made by the aggressor rat on the intruder was scored manually by a trained observer, including noting down the time of first attack (Table 2). A defeat was scored when the intruder rat submitted to the attack of the aggressor by lying down and exposing the ventral surface (abdomen; Figure 2C).

  7. This direct physical contact was allowed for a maximum of 15 min each session. The experimental rat was separated with the wire mesh enclosure prior to 15 min if any of the following criteria was met: (i) submission of the intruder; (ii) ten attacks with no submission; (iii) 5 min with no attack; or (iv) any attack that severely wounded the experimental rat. Thus, it was possible for a rat to be removed without demonstrating submission each day. Parameters can be modified if experimental design calls for daily defeat.

  8. PHASE 2 (Physical separation): The intruder was separated within the resident rat’s cage using a smaller wire mesh enclosure for an additional 15 min (Figure 1C, Figure 2C). The time of physical separation was noted.

    Note: The wire mesh enclosure used for physical separation was spacious enough not to cause any form of physical restraint to the movements of either the intruder rat inside or the resident rat outside the mesh enclosure. This was done in order to remove only the physical component of the social stress in Phase 2, without affecting the sensory (visual, auditory, and olfactory) components associated with the stressors (aggressor rats).

  9. All surfaces were thoroughly cleaned with 70% ethanol between experiments, including wire mesh enclosures.

  10. This same procedure was repeated for five consecutive days, once per day for each intruder rat. Resident-intruder pairings were cycled such that each intruder experienced a novel resident each day.

    Notes:

    a. Resident rats were limited to three daily sessions. After three daily sessions, or after extended periods of time (weeks to months), resident retired breeder rats tend to display less aggression. It has also been noted that resident rats tend to be less aggressive on the day of changes to a clean housing cage.

    b. If a rat were to get badly wounded during an attack (eye damaged, large wound) it would be removed and humanely euthanized. In some instances, a rat might receive a wound that is not severe (e.g. <2 cm to flank, without apparent muscle damage). If this occurs, the wound should be flushed with sterile saline followed by application of antibiotic+analgesic cream (e.g. Neosporin dual action cream). The rat should be monitored daily. If the wound does not close or otherwise show significant improvement within two days, the rat should be euthanized.

    c. If your endpoint includes immunological measures, you might consider removal of any rat that has been scratched, and pre-screening resident rats for those that do not tend to cause any wounds.



    Figure 2. Postures during attack and defeat.

    A. Examples of social interaction, followed by a common posture that signals an imminent attack: the resident rat rotates so it is flank-to-flank with the intruder and then lifts the hind leg adjacent to the intruder, to maintain position before it enters the next step. B. The next steps that escalate into the most common attack. The resident rat twists its body while keeping the hind leg close to the intruder, and then turns under the intruder. This flips the intruder over, with the resident on top. In other less common instances, the resident rat may initiate and continue an attack from the top position, for instance when both rats are in an upright boxing stance (not shown). After attacks, the intruder may avoid the resident rat. C. After the experience, the intruder rat shows a typical defeat posture. At this point in the protocol, the rats are separated with a wire mesh enclosure.


    Table 2. Example of a completed scoring sheet for a five day repeated social defeat protocol of three intruder rats (rats 1-3) that were exposed to a rotation of resident rats (rats 1-3, and 6-9).


    Instructions to make the wire mesh enclosure

    There are commercially available enclosures for pets that may be suitable. We have never tried those options and have instead made our enclosures from supplies available at most hardware stores. There are a range of suitable options for materials to make a wire mesh enclosure. We chose a PVC-coated steel mesh, because it is easy to cut, bend to the desired shape, and can be readily cleaned. A mesh opening of 0.5-1 inch is suitable for our purposes, because this is too small for the resident rat to effectively attack through. We chose a mesh of 16 gauge because of its durability and weight. The final dimensions are approximately 6 × 7 × 8 inches.


    Materials

    Heavy duty wire cutters

    Pliers

    Coated wire mesh (for example, item 9259T11 from McMaster-Carr; www.mcmaster.com)

    Zip ties (for example, item 6705K35 from McMaster-Carr; www.mcmaster.com)


    Steps:

    1. Cut the wire mesh to pattern (Figure 3).

    2. Bend where indicated to form an open cube.

    3. Fasten corners together with zip ties. Trim the ends of zip ties.



    Figure 3. Photograph of completed wire mesh enclosure.

    This enclosure has lasted eight years of use with minimal damage. A template and instructions are shown here.

Data analysis

Exclusion criteria: The intensity of the repeated stress experience can be varied, depending on the experimental needs. In our studies, rats had to exhibit at least one instance of defeat, demonstrated by a passive defeat posture (see Figure 2). Experimental rats were excluded if they received a severe injury (see Step 10 Notes, under the “Procedure” section). Resident rats were excluded if they exhibited two sessions without attacks, or if they consistently attacked with a severity that produced injury.


During the period of direct physical contact, every attack made by the aggressor rat on the intruder was scored manually by a trained observer, including noting down the time of first attack and the time of physical separation (Table 2). The following parameters were analyzed: number of attacks made by the aggressor on the intruder rats, whether rats submitted, and if so, time of submission by the intruder rat (i.e., latency to submit, which will be the same as the “Time separated” in our score sheet), and the number of rats that submitted daily. Conclusion

In this article, we discussed the protocol for the induction of RSDS in rats that we used in our study, showing that RSDS is able to induce robust immunological and behavioral changes in rats (Munshi et al., 2020). This builds on many prior studies that have successfully achieved social defeat in rats and mice.

Acknowledgments

The authors gratefully thank Matthew Anagnostopoulos and his team at the Biological Resource Facility at Rosalind Franklin University of Medicine and Science for taking care of the animals used in the study. The study was supported by the National Institutes of Health grants MH084970 and MH109484. The funding body had no role in the design of the study, collection and analysis of data, or decision to publish. The protocol is based on the original research article by Munshi et al. (2020) published in Brain, Behavior, and Immunity.

Competing interests

The authors declare no competing interests.

Ethics

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Rosalind Franklin University of Medicine and Science and were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

References

  1. Berton, O., McClung, C. A., Dileone, R. J., Krishnan, V., Renthal, W., Russo, S. J., Graham, D., Tsankova, N. M., Bolanos, C. A., Rios, M., Monteggia, L. M., Self, D. W. and Nestler, E. J. (2006). Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science 311(5762): 864-868.
  2. Bailey, M. T., Kinsey, S. G., Padgett, D. A., Sheridan, J. F. and Leblebicioglu, B. (2009). Social stress enhances IL-1beta and TNF-alpha production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells. Physiol Behav 98(3): 351-358.
  3. Chaudhury, D., Walsh, J. J., Friedman, A. K., Juarez, B., Ku, S. M., Koo, J. W., Ferguson, D., Tsai, H. C., Pomeranz, L., Christoffel, D. J., et al. (2013). Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature 493(7433): 532-536.
  4. Colyn, L., Venzala, E., Marco, S., Perez-Otano, I. and Tordera, R. M. (2019). Chronic social defeat stress induces sustained synaptic structural changes in the prefrontal cortex and amygdala. Behav Brain Res 373: 112079.
  5. 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.
  6. Jaisinghani, S. and Rosenkranz, J. A. (2015). Repeated social defeat stress enhances the anxiogenic effect of bright light on operant reward-seeking behavior in rats. Behav Brain Res 290: 172-179.
  7. Koolhaas, J. M., De Boer, S. F., De Rutter, A. J., Meerlo, P. and Sgoifo, A. (1997). Social stress in rats and mice. Acta Physiol Scand Suppl 640: 69-72.
  8. Liu, Y. Y., Zhou, X. Y., Yang, L. N., Wang, H. Y., Zhang, Y. Q., Pu, J. C., Liu, L. X., Gui, S. W., Zeng, L., Chen, J. J., et al. (2017). Social defeat stress causes depression-like behavior with metabolite changes in the prefrontal cortex of rats. PLoS One 12(4): e0176725.
  9. Munshi, S., Loh, M. K., Ferrara, N., DeJoseph, M. R., Ritger, A., Padival, M., Record, M. J., Urban, J. H. and Rosenkranz, J. A. (2020). Repeated stress induces a pro-inflammatory state, increases amygdala neuronal and microglial activation, and causes anxiety in adult male rats. Brain Behav Immun 84: 180-199.
  10. Powell, N. D., Bailey, M. T., Mays, J. W., Stiner-Jones, L. M., Hanke, M. L., Padgett, D. A. and Sheridan, J. F. (2009). Repeated social defeat activates dendritic cells and enhances Toll-like receptor dependent cytokine secretion. Brain Behav Immun 23(2): 225-231.
  11. Patel, D., Anilkumar, S., Chattarji, S. and Buwalda, B. (2018). Repeated social stress leads to contrasting patterns of structural plasticity in the amygdala and hippocampus. Behav Brain Res 347: 314-324.
  12. Riga, D., Theijs, J. T., De Vries, T. J., Smit, A. B. and Spijker, S. (2015). Social defeat-induced anhedonia: effects on operant sucrose-seeking behavior. Front Behav Neurosci 9: 195.
  13. Rygula, R., Abumaria, N., Flugge, G., Fuchs, E., Ruther, E. and Havemann-Reinecke, U. (2005). Anhedonia and motivational deficits in rats: impact of chronic social stress. Behav Brain Res 162(1): 127-134.
  14. Rygula, R., Abumaria, N., Domenici, E., Hiemke, C. and Fuchs, E. (2006). Effects of fluoxetine on behavioral deficits evoked by chronic social stress in rats. Behav Brain Res 174(1): 188-192.
  15. Stark, J. L., Avitsur, R., Hunzeker, J., Padgett, D. A. and Sheridan, J. F. (2002). Interleukin-6 and the development of social disruption-induced glucocorticoid resistance. J Neuroimmunol 124(1-2): 9-15.
  16. Tallerova A. V.,Kovalenko L. P., Tsorin I. B., Durney A. D. and Seredenin S. B. (2014). Effects of the Novel Anti-Asthenic Drug Ladasten on Behavior and T-Cell Subsets Alterations in a Social Defeat Animal Model of Depression.Pharmacol Pharm 5(1): 4-10.
  17. Tornatzky, W. and Miczek, K. A. (1993). Long-term impairment of autonomic circadian rhythms after brief intermittent social stress. Physiol Behav 53(5): 983-993.
  18. Tidey, J. W. and Miczek, K. A. (1997). Acquisition of cocaine self-administration after social stress: role of accumbens dopamine. Psychopharmacology (Berl) 130(3): 203-212.
  19. Tsankova, N. M., Berton, O., Renthal, W., Kumar, A., Neve, R. L. and Nestler, E. J. (2006). Sustained hippocampal chromatin regulation in a mouse model of depression and antidepressant action. Nat Neurosci 9(4): 519-525.
  20. Volkmann, P., Stephan, M., Krackow, S., Jensen, N. and Rossner, M. J. (2020). PsyCoP - A Platform for Systematic Semi-Automated Behavioral and Cognitive Profiling Reveals Gene and Environment Dependent Impairments of Tcf4 Transgenic Mice Subjected to Social Defeat. Front Behav Neurosci 14: 618180.
  21. Wohleb, E. S., Hanke, M. L., Corona, A. W., Powell, N. D., Stiner, L. M., Bailey, M. T., Nelson, R. J., Godbout, J. P. and Sheridan, J. F. (2011). β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. J Neurosci 31(17): 6277-6288.
  22. Wohleb, E. S., Powell, N. D., Godbout, J. P. and Sheridan, J. F. (2013). Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior. J Neurosci 33(34): 13820-13833.
  23. Yoshida, K., Drew, M. R., Kono, A., Mimura, M., Takata, N. and Tanaka, K. F. (2021). Chronic social defeat stress impairs goal-directed behavior through dysregulation of ventral hippocampal activity in male mice. Neuropsychopharmacology 46(9): 1606-1616.


简介

[摘要] 反复社交失败压力 (RSDS) 是啮齿动物慢性压力的一种模型。有几种社会失败程序的变体对小鼠产生了强大的影响,但很少有公布详细的协议来产生强大的压力和改变大鼠的免疫学特征。在本文中,我们描述了在成年雄性 Sprague-Dawley 大鼠中诱导 RSDS 的协议。使用常驻入侵者范式,压力的物理成分是由常驻侵略性退休饲养员 Long-Evans 大鼠对入侵者实验大鼠的直接攻击引起的。随后的威胁成分是由入侵者附近的攻击者的存在引起的,但它们之间存在物理隔离。该协议诱导的 RSDS 在实验大鼠中产生了强烈的免疫学和行为变化,这可以通过在开放场、社交互动和高架十字迷宫测试中的焦虑样行为的发展以及免疫参数的变化(Munshi 等等人,2020)。这种方法已被用作与行为学相关的压力源模型,这些模型足以影响与抑郁症和焦虑症患者受影响的神经回路相似的神经回路。

[背景]反复社交失败压力 (RSDS) 是啮齿动物慢性心理压力的稳健模型 (Rygula et al ., 2005; Berton et al ., 2006; Liu et al ., 2017)。它 导致强烈的抑郁样行为变化,包括焦虑、快感缺乏和社交回避(Rygula等人,2005 和 2006 年;Berton等人,2006 年)。与其他环境压力源不同的是,社会挫败压力的一个独特方面是受试者不会在反复的社会对抗中养成垂体-肾上腺轴活动的习惯(Tornatzky 和 Miczek,1993;Koolhaas等,1997),这与许多其他压力不同。重复的压力源,即使受试者产生行为压力反应(Tidey 和 Miczek,1997)。
此外,众所周知,RSDS 对影响啮齿动物焦虑行为的免疫参数具有强大的影响(Wohleb等人,2013 年)。此外,RSDS 通过大脑内皮细胞中的白细胞介素 1 型 1 型受体 (IL-1R1) 诱导焦虑样行为 (Wohleb et al ., 2011)。大脑中 RSDS 诱导的巨噬细胞转运是焦虑行为发展所必需的,而焦虑行为又依赖于小胶质细胞的激活和致敏单核细胞向大脑的募集 (Wohleb et al ., 2013)。从接受 RSDS 的小鼠中分离的骨髓细胞在免疫原脂多糖刺激 toll 样受体后,白细胞介素 (IL) 和其他细胞因子(如 IL-1β、TNF-α 和 IL-6)的产生增加(Stark等人, 2002;贝利等人,2009;鲍威尔等人,2009)。
通过检查其对边缘区域的影响,已利用社交失败程序来了解焦虑或抑郁行为的神经生物学。 RSDS 还被证明可以减少小鼠从腹侧被盖区 (VTA) 投射到内侧前额叶皮层 (mPFC) 的多巴胺能神经元的放电(Chaudhury等人,2013 年)。此外,已知这种压力会导致基底外侧杏仁核 (BLA)、海马体和 PFC 的持续神经元适应。例如,RSDS 导致 CA1 海马神经元的顶端树突棘密度降低,但在大鼠的 BLA 神经元中没有;它还导致 CA1 基底树突的树突萎缩,同时增加 BLA 锥体神经元的树突树枝状化(Patel等,2018)。一种类似的压力范式,称为慢性社交失败压力(CSDS),已被证明可以降低 PFC 锥体神经元顶端树突的脊柱密度,但在一个月后(长期)增加 BLA 树枝状树突;然而,它也在短期内增加了 BLA 星状神经元尖峰密度(Colyn等人,2019 年)。
与对开放场地和迷宫的影响相比,社交失败的某些变化可以优先对食欲动机行为产生影响(描述为快感缺乏/抑郁行为的啮齿动物模型;Riga等人,2015 年;吉田等人,2021 年)探索(描述为焦虑的啮齿动物模型)。在这种情况下,社会失败已显示出抗抑郁药物的一定程度的预测有效性(Tsankova等人,2006 年;Golden等人,2011 年)。最近在青春期转基因小鼠中研究了类似的社会失败压力范式,以通过环境相互作用探索基因型,从而在“二次打击”模型中导致精神疾病表型的发展。例如,过度表达Tcf4的小鼠在暴露于社交失败压力时会出现认知障碍和新奇诱导的多动症(Volkmann等人,2021)。社交失败压力也被用于小鼠测试精神药物的药理作用,以研究压力引起的免疫失调。例如,一项研究表明,抗虚弱药物溴烷 (Ladasten) 通过阻止回避行为的发展以及通过改善压力后的运动活动来减少抑郁样行为。同样,Ladasten 还阻止了应激诱导的 CD4/CD8 T 细胞向 T 细胞毒性细胞的转变,并使它们在小鼠胸腺、脾脏和血液中的比例正常化(Tallerova等,2014)。
虽然已经公布了小鼠社交失败的协议,但大鼠的社交失败,特别是反复的社交失败,有其自身的一系列挑战。很少有详细的协议可以在大鼠中反复进行社交失败程序,这些程序可以可靠地产生压力表型并改变外周免疫功能。本文的一个目的就是满足这一需求。 RSDS 模型利用持续的社会动态,而不是强加不寻常的情况,这提供了一定程度的面子有效性,并且可以提供比自然压力源更多的可翻译信息,以了解个体之间的社会压力引起的一些生理和行为异常.在我们最初的研究(Munshi等人,2020 年)中,我们按照先前发布的方案(Jaisinghani 和 Rosenkranz,2015 年)在成年雄性 Sprague-Dawley 大鼠中使用了 RSDS 模型。这已经从早期发表的使用压力的社会失败模型(Berton et al ., 2006)的研究中改编和修改。

受试者和住房:成年雄性 Sprague-Dawley 大鼠 (Envigo, Indianapolis, IN) 在出生后第 59-63 天获得,每笼 2-3 只饲养在罗莎琳德富兰克林医学与科学大学的气候控制设施中,随意获得标准大鼠食物和水。在动物设施中适应至少四天后,对大鼠进行 RSDS 或对照处理。 Sprague-Dawley 大鼠的体重在实验开始时为 300-350 克。宿舍间的灯按照相反的 12 小时明暗时间表(熄灯时间:07:00-19:00)。在典型的实验室条件下,Sprague-Dawley 大鼠具有相当的社交能力,很少攻击其他大鼠,除了打架或仍在哺乳的水坝。因此,需要一个替代方案来诱导失败。成年雄性退休饲养员 Long-Evans 大鼠(Envigo,Indianapolis,IN)是单圈饲养的,用于诱导 Sprague-Dawley 大鼠的社会失败压力。大鼠被随机分配到实验组,并在多个队列中进行实验。社交失败实验是在动物设施内的一个单独的程序室中进行的,该程序室专门用于压力程序。这个单独的房间距离宿舍大约8米。实验是在大鼠昼夜循环的黑暗阶段,即上午 9 点至下午 2 点之间进行的。居民和入侵者没有被关在同一个笼子里,就像老鼠的一些社交失败程序那样。在我们的程序中,居民和入侵者仅在每日一次的压力会议期间进行互动。
由于这些实验涉及压力,因此与我们的机构兽医协商建立超越标准人道终点的人道排除标准尤为重要。每天监测大鼠。这包括每天对大鼠称重、观察行为(嗜睡、极端回避实验者)和观察外观(外套外观、卟啉的存在)。如果任何大鼠表现出体重减轻 > 15%、持续的卟啉染色、嗜睡或不梳理毛发的迹象(毛茸茸的毛发),则将其从研究中移除。 Long-Evans 大鼠也在社交失败程序的过程中进行了相同的测量(观察行为和外观)。

关键字:侵略鼠, 家笼, 侵略鼠, 心理压力, 社交失败, 提交

材料和试剂

 

1. 干净的纸巾

2. 入侵者(实验性)成年雄性 Sprague-Dawley 大鼠

3. 居民(攻击者)成年雄性退休饲养员 Long-Evans 大鼠

4. 评分表(表 1

5. 70% 乙醇

6. 用于设备的消毒湿巾(例如,Caviwipes 表面消毒湿巾,Metrex item# 13-1100

 

1. 评分表。这是一个标准的计分表,对社交失败程序很有用。提交后,或者如果满足任何标准 (ac),则第 1 阶段会话结束,入侵大鼠被放置在金属丝网罩中。第 2 阶段会话(由金属丝网罩隔开)在 15 分钟后结束。有关详细信息,请参阅下面的完整表格(表 2)。

 

 

设备

 

1. 居民鼠笼:消毒木片床上用品,21” × 11.5” × 8” 高,透明聚碳酸酯,金属丝顶

2. 大鼠运输笼:消毒木片床上用品,12” × 6.5” × 5” 高,透明聚碳酸酯,微型隔离器顶部

3. 较小的金属丝网外壳(图 3):大约 6” × 7” × 8” 高,3/4” 方形网格,PVC 涂层

4. 带时间的数字秒表

 

程序

 

1. 居民入侵者范式的 RSDS 是啮齿动物慢性心理压力的稳健模型(Rygula等人2005Berton等人2006Liu等人2017)。在我们的研究中,老鼠的笼子被随机分配到压力组和对照组。

注意:大多数 Long-Evans 退休饲养员在我们的条件下都具有侵略性,但有些不是。您可以在实验之前筛选这些老鼠,使用下面描述的相同的常驻入侵者方法,根据您的需要选择或多或少具有攻击性的老鼠。如果需要更积极的居民行为,预筛选标准可能包括攻击的短延迟和会话第一分钟内的多次攻击。在我们的实验中,退休的饲养员在几周和几个月的时间里表现出减少的攻击。如果退休的饲养员没有表现出攻击,它可能会从激进的居民轮换中移除。

2. 在实验当天早上,将常驻攻击者 Long-Evans 大鼠的笼子(每个笼子包含一只 Long-Evans 大鼠)运送到动物设施内光线昏暗且没有噪音的手术室。让它们不受干扰地在环境中适应 15-30 分钟。

注意:根据我们的经验,单个实验者可以同时执行此程序,最多可与 3 个常驻入侵者交互(即程序室中的 3 个常驻笼子)。一个人很难观察到更多。

3. 实验性 Sprague-Dawley 大鼠每天在其饲养室中称重,并记录它们的体重。还注意到动物状况,注意眼睛、鼻子或皮毛周围的卟啉。

4. 称重后,将 Sprague-Dawley 大鼠置于干净的运输笼中。

a. 对照大鼠(饲养在一起)在运输笼中停留 30 分钟,然后将它们放回其家笼中。

b. 应激组的大鼠被运送到与对照组分开的干净运输笼中的手术室。

5. 阶段 1(直接身体接触):将个体 Sprague-Dawley 大鼠转移到手术室中常驻 Long-Evans 大鼠的笼子中,并紧紧固定笼子的盖子。这使得入侵者大鼠与常驻大鼠保持直接的身体接触(图 1)。记录了这一事件的时间。在整个交互过程中连续观察大鼠。

一组大鼠的所有评分均由一名训练有素的实验者进行。实验者被定位在允许清晰观察大鼠和快速干预的距离处,但足够远以至于大鼠不会将注意力指向实验者(约1-2米)。这些措施可以由一名实验者可靠地获得,因此我们没有定期进行视频会议。

 

 

1. 设置常驻入侵者程序。

A.带有金属丝网罩(绿色)的居民笼子的照片(左)。还显示了居民在场(右)的比例。 B.压力程序的第一阶段,当居民和入侵者可以有直接的身体接触时。 C.应力程序的第 2 阶段,当居民和入侵者被金属丝网罩隔开时。

 

6. 在直接身体接触期间,攻击者对入侵者的每次攻击均由训练有素的观察员手动评分,包括记下第一次攻击的时间(表 2)。当入侵者大鼠通过躺下并暴露腹面(腹部;图 2C)来屈服于攻击者的攻击时,就获得了失败。

7. 这种直接的身体接触每次最多允许 15 分钟。如果满足以下任一标准,则实验大鼠在 15 分钟前用金属丝网罩隔开 (i) 入侵者提交; (ii) 十次没有屈服的攻击; (iii) 5 分钟无攻击; (iv) 任何严重伤害实验大鼠的攻击。因此,有可能在没有每天表现出提交的情况下将老鼠移走。如果实验设计要求每日失败,则可以修改参数。

8.  2 阶段(物理分离):入侵者在常驻大鼠笼内使用较小的金属丝网罩隔开15 分钟(图 1C,图 2C)。记录物理分离的时间。

注意:用于物理分离的金属丝网罩足够宽敞,不会对网罩内的入侵大鼠或网罩外的常驻大鼠的运动造成任何形式的物理限制。这样做是为了在第 2 阶段仅消除社会压力的物理成分,而不影响与压力源(攻击性大鼠)相关的感觉(视觉、听觉和嗅觉)成分。

9. 在实验之间用 70% 乙醇彻底清洁所有表面,包括金属丝网罩。

10. 连续五天重复相同的程序,每只入侵大鼠每天一次。居民-入侵者配对是循环的,这样每个入侵者每天都会经历一个新的居民。

笔记:

a. 常驻大鼠仅限于每天 3 次。在每天三个会话后,或经过较长时间(数周至数月)后,居民退休的饲养鼠往往表现出较少的攻击性。还注意到,在更换干净的笼子的当天,常驻老鼠的攻击性往往较小。

b.如果一只老鼠在一次攻击中受了重伤(眼睛受损,大伤口),它将被移除并人道地实施安乐死。在某些情况下,大鼠可能会受到不严重的伤口(例如,侧面小于 2 厘米,没有明显的肌肉损伤)。如果发生这种情况,应该用无菌盐水冲洗伤口,然后使用抗生素+镇痛药膏(例如新孢霉素双效药膏)。应每天监测大鼠。如果伤口在两天内没有闭合或以其他方式显示出显着改善,则应对大鼠实施安乐死。

c. 如果您的终点包括免疫学措施,您可能会考虑移除任何被划伤的老鼠,并对那些不会造成任何伤口的老鼠进行预筛选。

 

 

2. 攻击和失败期间的姿势。 

A.社交互动的例子,然后是一个常见的姿势,表明即将发动攻击:常驻大鼠旋转,使其与入侵者侧翼并列,然后抬起靠近入侵者的后腿,以在它进入之前保持位置下一步。 B.升级为最常见攻击的后续步骤。常驻老鼠在保持后腿靠近入侵者的同时扭转身体,然后在入侵者下方转身。这将入侵者翻转过来,居民在上面。在其他不太常见的情况下,常驻大鼠可能会从顶部位置开始并继续攻击,例如当两只大鼠都处于直立拳击姿势时(未显示)。攻击后,入侵者可能会避开常驻老鼠。 C.体验后,入侵鼠表现出典型的失败姿势。在协议的这一点上,老鼠被一个金属丝网罩隔开。

 

 

2.三只入侵大鼠 (大鼠 1-3) 的五天重复社会失败协议的完整评分表示例,这些入侵大鼠 (大鼠 1-3) 暴露于常驻大鼠 (大鼠 1-3 6-9) 的轮换中。

 

 

 

制作金属丝网罩的说明

有市售的宠物围栏可能是合适的。我们从未尝试过这些选项,而是使用大多数五金店提供的耗材制造我们的外壳。制作金属丝网罩的材料有多种合适的选择。我们选择了 PVC 涂层钢网,因为它易于切割、弯曲成所需的形状,并且易于清洁。 0.5 - 1 英寸的网孔适合我们的目的,因为这对于常驻老鼠来说太小而无法有效攻击。由于其耐用性和重量,我们选择了 16 号网眼。最终尺寸约为 6 × 7 × 8 英寸。

 

材料

重型钢丝钳

涂层金属丝网(例如,McMaster-Carr 的产品 9259T11www.mcmaster.com

拉链(例如,来自 McMaster-Carr 6705K35 项; www.mcmaster.com

 

脚步:

1. 将金属丝网切割成图案(图 3)。

2. 在指示处弯曲以形成一个开放的立方体。

3. 用拉链系紧边角。修剪拉链的末端。

 

 

3.完成的金属丝网罩的照片。

这个外壳已经使用了八年,并且损坏最小。此处显示了模板和说明。

 

数据分析

 

排除标准:重复压力体验的强度可以根据实验需要而变化。在我们的研究中,老鼠必须表现出至少一次失败,表现为被动失败姿势(见图 2)。如果实验大鼠受到严重伤害,则被排除在外(参见程序部分下的步骤 10 注释)。如果常驻大鼠表现出两次没有攻击的情况,或者如果它们持续以产生伤害的严重程度攻击,则将它们排除在外。

入侵者的每次攻击都由训练有素的观察者手动评分,包括记下第一次攻击的时间和物理分离的时间(表 2)。分析了以下参数:攻击者对入侵大鼠的攻击次数,大鼠是否提交,如果是,入侵者大鼠提交的时间(,延迟提交,这将与时间相同分开在我们的评分表中),以及每天提交的老鼠数量。


结论

在本文中,我们讨论了我们在研究中使用的大鼠 RSDS 诱导方案,表明 RSDS 能够诱导大鼠强烈的免疫和行为变化(Munshi等人2020)。这是建立在许多先前的研究基础上的,这些研究已经成功地在大鼠和小鼠身上实现了社交失败。

 

 

致谢

 

作者非常感谢罗莎琳德富兰克林医科大学生物资源设施的 Matthew Anagnostopoulos 和他的团队照顾研究中使用的动物。该研究得到了美国国立卫生研究院 MH084970 MH109484 的资助。资助机构在研究设计、数据收集和分析或发表决定中没有任何作用。该协议基于 Munshi等人的原始研究文章 (2020) 发表在《大脑、行为和免疫》上。

 

利益争夺

 

作者声明没有竞争利益。 

 

 

伦理

 

所有实验均经罗莎琳德富兰克林医科大学机构动物护理和使用委员会 (IACUC) 批准,并按照《实验动物护理和使用指南》(国家研究委员会,2011 年)进行。

 

 

参考

 

1. Berton, O., McClung, CA, Dileone, RJ, Krishnan, V., Renthal, W., Russo, SJ, Graham, D., Tsankova, NM, Bolanos, CA, Rios, M., Monteggia, LM, Self , DW Nestler, EJ (2006)BDNF 在社会挫败压力中的中脑边缘多巴胺通路中的重要作用。科学3115762):864-868

2. Bailey, MT, Kinsey, SG, Padgett, DA, Sheridan, JF Leblebicioglu, B. (2009)社会压力增强了牙龈卟啉单胞菌脂多糖刺激的 CD11b +细胞产生的 IL-1beta TNF-alpha 。生理行为 983):351-358

3. Chaudhury, D., Walsh, JJ, Friedman, AK, Juarez, B., Ku, SM, Koo, JW, Ferguson, D., Tsai, HC, Pomeranz, L., Christoffel, DJ2013)。通过控制中脑多巴胺神经元快速调节抑郁相关行为。 自然4937433):532-536

4. Colyn, L.Venzala, E.Marco, S.Perez-Otano, I. Tordera, RM (2019)慢性社交失败压力会导致前额叶皮层和杏仁核持续的突触结构变化。行为大脑研究373112079

5. Golden, SA, Covington, HE, 3rd, Berton, O. Russo, SJ (2011)小鼠反复社交失败压力的标准化协议。 国家协议68):1183-1191

6. Jaisinghani, S. Rosenkranz, JA (2015)反复的社会挫败压力增强了强光对大鼠操作性寻求奖励行为的焦虑作用。行为大脑研究290172-179

7. Koolhaas, JM, De Boer, SF, De Rutter, AJ, Meerlo, P. Sgoifo, A. (1997)大鼠和小鼠的社会压力。 Acta Physiol Scand Suppl 64069-72

8. Liu, YY, Zhou, XY, Yang, LN, Wang, HY, Zhang, YQ, Pu, JC, Liu, LX, Gui, SW, Zeng, L., Chen, JJ, et al . 2017)。社交失败压力会导致大鼠前额叶皮层代谢物发生变化的抑郁样行为。公共科学图书馆一号12(4)e0176725

9. Munshi, S.Loh, MKFerrara, N.DeJoseph, MRRitger, A.Padival, M.Record, MJUrban, JH Rosenkranz, JA (2020)反复的压力会诱导促炎状态,增加杏仁核神经元和小胶质细胞的激活,并导致成年雄性大鼠焦虑。脑行为免疫84180-199

10. Powell, ND, Bailey, MT, Mays, JW, Stiner-Jones, LM, Hanke, ML, Padgett, DA Sheridan, JF (2009)反复的社交失败会激活树突状细胞并增强 Toll 样受体依赖性细胞因子分泌脑行为免疫232):225-231

11. Patel, D., Anilkumar, S., Chattarji, S. Buwalda, B. (2018)反复的社会压力导致杏仁核和海马体结构可塑性的对比模式。 行为大脑研究347314-324

12. Riga, D., Theijs, JT, De Vries, TJ, Smit, AB Spijker, S. (2015)社会失败引起的快感缺乏:对操作性寻求蔗糖行为的影响。 前行为神经科学9195

13. Rygula, R.Abumaria, N.Flugge, G.Fuchs, E.Ruther, E. Havemann-Reinecke, U. (2005)大鼠的快感缺失和动机缺陷:慢性社会压力的影响。行为大脑研究1621):127-134

14. Rygula, R.Abumaria, N.Domenici, E.Hiemke, C. Fuchs, E. (2006)氟西汀对大鼠慢性社会压力引起的行为缺陷的影响行为大脑研究1741):188-192

15. Stark, JL, Avitsur, R., Hunzeker, J., Padgett, DA Sheridan, JF (2002)白细胞介素 6 和社会混乱诱导的糖皮质激素抵抗的发展。 神经免疫杂志1241-2):9-15

16. Tallerova AVKovalenko LPTsorin IBDurney ADSeredenin SB (2014)新型抗虚弱药物 Ladasten 对抑郁症社会失败动物模型中行为和 T 细胞亚群改变的影响。 Pharmacol 制药 51):4-10

17. Tornatzky, W. Miczek, KA (1993)短暂的间歇性社会压力后自主昼夜节律的长期损害。生理学行为 535):983-993

18. Tidey, JW Miczek, KA (1997)社会压力后可卡因自我管理的获得:伏隔核多巴胺的作用。精神药理学 (Berl) 130(3): 203-212

19. Tsankova, NM, Berton, O., Renthal, W., Kumar, A., Neve, RL Nestler, EJ (2006)在抑郁症和抗抑郁作用的小鼠模型中持续的海马染色质调节。 Nat Neurosci 94):519-525

20. Volkmann, P.Stephan, M.Krackow, S.Jensen, N. Rossner, MJ (2020)PsyCoP - 系统半自动行为和认知分析平台揭示了 Tcf4 转基因小鼠遭受社会失败的基因和环境依赖性损伤。 前行为神经科学14618180

21. Wohleb, ES, Hanke, ML, Corona, AW, Powell, ND, Stiner, LM, Bailey, MT, Nelson, RJ, Godbout, JP Sheridan, JF (2011)β-肾上腺素受体拮抗作用可防止反复社交失败引起的焦虑样行为和小胶质细胞反应。神经科学杂志 3117):6277-6288

22. Wohleb, ES, Powell, ND, Godbout, JP Sheridan, JF (2013)压力诱导的骨髓来源的单核细胞向大脑的募集促进了焦虑样行为神经科学杂志 3334):13820-13833

Yoshida, K.Drew, MRKono, A.Mimura, M.Takata, N. Tanaka, KF (2021)慢性社交失败压力通过雄性小鼠腹侧海马活动的失调损害目标导向行为。 神经精神药理学469):1606-1616

登录/注册账号可免费阅读全文
  • 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: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用:Munshi, S., Ritger, A. and Rosenkranz, J. A. (2022). Induction of Repeated Social Defeat Stress in Rats . Bio-protocol 12(3): e4306. DOI: 10.21769/BioProtoc.4306.
提问与回复

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。