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Method Article

A Multiple Integrated Social Stress Model for Psychiatric Disorders in Female C57BL/6J Mice

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DOI:

10.3791/67117

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July 15th, 2025

 ,  ,  ,  , 
1Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, 2Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, 3NMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, Xuzhou Medical University

In This Article

Summary

By integrating four daily social stressors, the present study describes a multiple integrated social stress (MISS) model for psychiatric disorders in female C57BL/6J mice. After ten days of repeated MISS exposure, the mice developed depressive- and anxiety-like phenotypes, thus providing a natural, etiology-based animal model for studying psychiatric disorders in female subjects.

Abstract

Despite females accounting for the majority of individuals suffering from psychiatric disorders, preclinical studies have almost exclusively focused on male subjects, partly due to the lack of ideal female animal paradigms. Developing effective models for studying psychiatric disorders in female animals remains a long-standing scientific challenge in the life sciences. A "Multiple Integrated Social Stress (MISS) model" in C57BL/6J female mice was recently established by simulating and integrating social risk factors that contribute to the development of psychiatric disorders. To establish this MISS paradigm, female C57BL/6J mice were randomly subjected each day, for ten consecutive days, to a sequence of stressors: social competition failure in the tube test, modified vicarious social defeat stress, inescapable overcrowding stress, and subsequent social isolation. Compared to naïve mice, MISS-exposed mice exhibited depressive- and anxious-like phenotypes, as measured by the sucrose preference, tail suspension, open field, and elevated plus maze tests. This paradigm offers a valuable tool for investigating the neurobiological mechanisms underlying depression and anxiety in females, particularly those with ambiguous etiology and complex symptomatology.

Introduction

Hundreds of millions of people are living with psychiatric disorders, with anxiety and depressive disorders being the most common. According to the World Health Organization, more than 280 million and 301 million people worldwide were living with depression and anxiety, respectively, in 20191. Due to the COVID-19 pandemic, the number of people affected by anxiety and depressive disorders increased significantly-by 26% and 28%, respectively-within just one year. Among those affected, the lifetime prevalence of psychiatric disorders in women is nearly twice as high as in men2, with women exhibiting more severe symptoms, greater functional impairment, atypical depressive phenotypes, more frequent relapses, and lower responsiveness to medications3,4,5.

Although sex differences in many aspects of psychiatric disorders are well-documented6,7,8, most preclinical research has predominantly focused on male subjects, primarily due to the lack of appropriate female animal models9,10. As a result, the pathogenesis of these disorders in women remains poorly understood. This emphasizes the urgent need to develop animal models that include female subjects, which would facilitate the investigation of neurobiological mechanisms underlying psychiatric disorders in females.

An increasing number of social stress models for female mice have been developed based on the repeated social defeat stress (RSDS) paradigm11,12,13,14. For instance, two recently established RSDS models in female mice involved artificially enhancing male aggression through the application of male urine to female mice and chemogenetic activation of the ventrolateral subdivision of the ventromedial hypothalamus in male aggressors11,12,13. However, it has been argued that these induced aggressive behaviors are not naturally occurring and may not represent relevant stressors for female mice10. In addition, pregnancy resulting from mounting behavior complicates subsequent mechanistic studies.

Another RSDS-based modified model is the vicarious social defeat stress paradigm, in which female mice visually witness inter-male social defeat12. Female-female social defeat protocols have also been developed in various rodent strains14,15,16. Despite some concerns, the social stressors used in these latter two models appear to be more etiologically relevant to human psychiatric disorders14. An ideal depression model requires construct (etiologic) validity, meaning that the stress-inducing methods used in the model must closely reflect the real-life causes of the disorder.

The etiology of psychiatric disorders is believed to be multifactorial, involving biological, genetic, environmental, and psychosocial factors. Manipulating these risk factors in laboratory animals is essential for developing relevant models for mechanistic research and drug screening. Both social and non-social animal models that simulate one or more stressors can replicate core symptoms and neurobiological alterations associated with human psychiatric disorders. For example, the chronic mild stress (CMS) model induces stable and effective neuropathological and depressive-like behavioral phenotypes by randomly alternating combinations of multiple physical stressors (e.g., wet bedding, tail suspension, tail clamping, fasting)17,18,19,20,21,22,23. These findings suggest that repeated modeling of multiple risk factors is a viable strategy for constructing paradigms to study psychiatric disorders. The chronic social defeat stress (CSDS) model is one of the most widely used paradigms for depression that simulates social stressors24,25,26. Although this model is limited to mimicking a single type of stressor, its etiological relevance has led to significant progress in translational research. For instance, CSDS-based studies have identified potassium (K+) channels on dopaminergic neurons in the ventral tegmental area as key targets mediating resilience to depression, and have highlighted KCNQ-type K+ channel openers-such as retigabine (ezogabine)-as promising antidepressant candidates in both depressive-like mice and patients with depression27,28,29,30,31. As a result, several seminal studies have attempted to modify the CSDS paradigm to model female depression by inducing female-directed aggression11,12. The evidence above supports the notion that integrating multiple social stressors with repeated exposure represents an important direction for developing psychiatric models in female animals.

In female C57BL/6J mice, a 10-day multiple integrated social stress (MISS) paradigm was recently developed to model psychiatric disorders, demonstrating reliable etiologic, face, construct, and predictive validities32. To establish the MISS model, female C57BL/6J mice were randomly subjected each day for 10 consecutive days to four sequential stressors: failure in social competition (tube test), modified vicarious social defeat stress, inescapable overcrowding stress, and subsequent social isolation32. This study provides a detailed protocol for modeling the MISS paradigm.

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Protocol

All experimental procedures were approved by the Xuzhou Medical University Animal Care and Use Committee (Approval No. 202207S127) and were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. C57BL/6J female mice (7-8 weeks old for subject mice; 10-14 weeks old for winner candidates), C57BL/6J male mice (7-8 weeks old for aggressive CD1 screening), and CD1 retired breeder mice used in these experiments were obtained from a commercial source. All mice were housed under a 12-h light/dark cycle with food and water provided ad libitum33. Baseline behavioral measurements were taken-although not necessarily for every cohort-prior to initiation of the MISS paradigm, to ensure that subsequent behavioral outcomes were attributable to stress exposure rather than individual variability. An overview of the full protocol is presented in Figure 1, and the completed experimental setups are shown in Figure 2. Details of the reagents and equipment used in this study are provided in the Table of Materials.

1. Habituation

  1. Habituation to the environment
    1. Designate a separate room exclusively for the MISS protocol, maintaining a constant temperature (23 °C ± 2 °C), humidity (50%-60%), and light intensity (15-20 lux) on a 12 h light-dark cycle.
      NOTE: Unless otherwise specified, house mice in this room throughout the entire modeling period.
    2. Prepare standard mouse cages (L × W × H: 37 cm × 16 cm × 12 cm) with steel-wire lids and corncob bedding.
    3. Use C57BL/6J female mice aged 7-8 weeks as subject mice. Randomly assign the mice to naïve and MISS groups, and house them at five mice per cage.
      NOTE: Select female mice with comparable age and body weight for the experiment.
    4. Use C57BL/6J female mice aged 10-14 weeks as candidates for winner screening30. House these mice in groups of four per cage to establish stable social ranks.
    5. Use male CD1 retired breeder mice as aggressor candidates. House CD1 mice individually to establish territoriality and prevent fighting. Use cohort-reared C57BL/6J male mice (5 mice per cage, aged 7-8 weeks) as intruders for aggressive CD1 screening.
    6. Allow all mice to habituate and recover from transport stress for 7 days after purchase (Figure 1A1).
    7. House all mice with food and water available ad libitum.
  2. Habituation to tubes and handling
    1. Place a short acrylic tube (Figure 2A; 10 cm long, 26 mm internal diameter) in each cage of female subject mice and winner candidates. Allow mice to acclimate to the tubes and learn to pass through them for 3 days.
      NOTE: The tube's diameter should be just sufficient to permit one adult mouse to pass through smoothly without turning around.
    2. Handle female subject mice and winner candidates for 1-2 min/day for 3 days to reduce stress related to unfamiliar experimenters (Figure 1A2).
    3. At the end of the habituation period, mark each mouse's tail with a red or black oil-based marker.
      NOTE: Check the markings regularly and reapply if necessary.

2. Training for tube competition, winner screening, and CD1 aggressor screening

  1. Training for tube competition
    NOTE: For details on this procedure, refer to previous reports32,34.
    1. Prepare female subject mice, female winner candidates, transparent acrylic tubes (Figure 2B; 30 cm long, 26 mm internal diameter), plastic sticks, 75% ethanol and paper towels. Clean the table, tubes, and plastic sticks with 75% ethanol to eliminate odor cues.
    2. Gently grasp the tail of each female subject mouse or winner candidate and place the mouse on the table. Allow it to explore freely for approximately 1 min for environmental acclimatization.
    3. Hold the mouse by the tail and place it at one end of the tube.
      NOTE: Ensure the mouse's head faces the end of the tube, with its front paws touching the table, to minimize stress and facilitate entry.
    4. Release the tail once the mouse enters the tube and allow it to pass through voluntarily.
      NOTE: Use a plastic stick to gently touch the tail if the mouse stops or attempts to reverse.
    5. Place the mouse at the opposite end of the tube and repeat step 2.1.4. Count steps 2.1.3 - 2.1.5 as one cycle.
    6. Repeat steps 2.1.3 and 2.1.5 for two cycles (Figure 1B1).
    7. Return the mouse to its home cage. Clean the tube with 75% ethanol to remove odors, urine, and feces.
      NOTE: Ensure the tube is clean, dry, and free from residual alcohol odor before use with the next mouse.
    8. Repeat steps 2.1.2-2.1.7 for all female mice for 3 consecutive days, ensuring each mouse becomes accustomed to passing through the tube.
  2. Winner screening
    NOTE: For details on this procedure, refer to previous reports32,34.
    1. Prepare female winner candidates, tubes (30 cm long, 26 mm internal diameter), plastic sticks, 75% ethanol, paper towels, and a timer. Clean and dry the equipment to minimize odor cues.
    2. Before screening, retrain each mouse to pass through the tube for one cycle. Draw a visible middle line on the tube.
    3. Hold both mice by their tails and place them at opposite ends of the tube. Allow them to enter and meet at the midpoint, then simultaneously release them and start the timer.
      NOTE: Use mice from the same cage for each competition.
    4. Identify the "winner" as the mouse that pushes the other out of the tube; award it 1 point. The "loser" receives 0 points.
      NOTE: Use a plastic stick to prevent the winner from retreating. If both mice freeze and do not retreat within 1 min, gently touch each mouse's tail with plastic sticks to prompt action.
    5. Reverse the mice's starting positions and repeat steps 2.2.3-2.2.4, scoring the outcome again (Figure 1B2).
    6. Return both mice to their home cage and clean the tube thoroughly with 75% ethanol.
    7. Use a round-robin format to match each mouse with all other cage mates, repeating steps 2.2.3-2.2.5 for each pair.
      NOTE: This approach ensures comprehensive and fair ranking.
    8. Rank mice based on total scores. Select the highest-scoring mouse in each cage as the "winner" for subsequent experiments.
  3. CD1 aggressor screening
    NOTE: For details on this procedure, refer to the previous report35.
    1. Prepare CD1 retired breeder mice, C57BL/6J male mice (intruders), and a timer.
    2. Place a C57BL/6J male mouse directly into the home cage of a CD1 male (resident aggressor) for 3 min.
      NOTE: The intruder cannot escape during the session.
    3. Record the latency to the first attack, the number of attacks, and the duration of aggressive behaviors within the 3-min period as indicators of aggressiveness.
    4. Repeat steps 2.3.2-2.3.3 once daily for 3 consecutive days using different intruder mice each day (Figure 1B3).
    5. Select CD1 mice as aggressors for further experiments based on two criteria: (1) they must have attacked in at least two of the three sessions, and (2) their attack latency must be less than 1 min in each session.

3. Modeling

NOTE: This step involves random exposure to social competition failure in the tube test, modified vicarious social defeat stress, and inescapable overcrowding stress, followed by social isolation, for 10 consecutive days.

  1. Social competition failure
    NOTE: For details, refer to previous reports32,34.
    1. Prepare female subject mice, screened "winner" mice, tubes (30 cm long, 26 mm internal diameter), plastic sticks, 75% ethanol, paper towels, and a timer.
    2. Re-house female subject mice individually (1 mouse per cage) and house naïve mice in groups (5 mice per cage).
      NOTE: Move the naïve mice to novel group cages at the start of modeling. Changing to a new cage is not required during the modeling period.
    3. Clean the table, tubes, and plastic sticks with 75% ethanol to eliminate odor cues.
    4. Carefully remove mice from their home cages. Place a MISS mouse and a "winner" mouse at opposite ends of the tube so that they walk forward and meet at the midpoint. The MISS mouse will be pushed out of the tube by the "winner."
      NOTE: The larger size, higher body weight, and dominant social rank of the "winner" ensure that the experimental MISS mouse almost always loses the tube competition. If the mice remain immobile for more than 30 s, gently touch the tail of the winner with a plastic stick to encourage pushing.
    5. Switch the positions of the two mice and repeat step 3.1.4. Count steps 3.1.4 and 3.1.5 as one cycle.
    6. Return the MISS and "winner" mice to their home cages. Clean the table, tube, and plastic stick to remove odors.
    7. Introduce a novel winner mouse and repeat steps 3.1.4-3.1.5 with the same MISS mouse. Each MISS mouse thus undergoes two consecutive cycles of failed tube competition (Figure 1C1, Figure 2E).
    8. Repeat steps 3.1.4-3.1.7 for all MISS mice for 10 consecutive days.
  2. Modified vicarious social defeat stress
    1. Prepare screened aggressive CD1 retired breeder mice (singly housed), male C57BL/6J mice, female MISS mice, and perforated acrylic covers (Figure 2C; L × W × H: 8 cm × 8 cm × 5.5 cm) with holes (5 × 5 in the top panel, 3 × 5 in the sidewalls; each hole 0.5 cm diameter).
    2. Place a female MISS mouse inside a CD1 aggressor's cage, confined beneath a perforated acrylic cover, to allow vicarious observation of aggressive interactions.
      NOTE: Monitor for potential tipping of the acrylic cover by either the CD1 or MISS mouse. Reducing corn cob bedding in the CD1 cages helps prevent tipping.
    3. Introduce a male C57BL/6J intruder mouse into the cage to be physically attacked by the CD1 mouse for 10 min. The female MISS mouse observes the interaction through the perforated cover (Figure 1C2, Figure 2F).
      NOTE: If the intruder mouse is severely injured, immediately terminate the session and treat the animal. Using small corn cob pellets enhances the immersive experience for the MISS mouse by amplifying bedding displacement during fights.
    4. After 10 min, remove the male intruder and allow the female MISS mouse an additional 5 min of sensory exposure to the CD1 mouse.
    5. Repeat steps 3.2.3-3.2.4 two more times with different male intruders.
    6. Return the MISS mouse to its home cage. Monitor and treat any injuries in the male intruders.
    7. Clean the perforated acrylic covers and sweep up any spilled bedding.
    8. Repeat steps 3.2.2-3.2.5 using a novel CD1 retired breeder for 10 consecutive days.
  3. Inescapable overcrowding stress
    1. Prepare female MISS mice, a white opaque sleeve (Figure 2D; L × W × H: 14 cm × 10 cm × 20 cm), standard mouse cages with corn cob bedding, 75% ethanol, paper towels, and a timer.
    2. Place the opaque sleeve inside a standard cage with bedding.
    3. Introduce 12-15 female mice (either all MISS mice or mixed with other females) into the sleeve for 30 min daily (Figure 1C3, Figure 2G).
      NOTE: Adjust the number of mice according to their size to ensure crowding without trampling. Prevent escape by covering the sleeve with a clear perforated lid if needed.
    4. Return the mice to their home cages and clean the sleeve with 75% ethanol.
    5. Repeat step 3.3.3 for 10 consecutive days.
  4. Social isolation
    1. Prepare female MISS mice and standard mouse cages with corn cob bedding.
    2. House MISS mice individually when they are not undergoing social competition failure, vicarious defeat stress, or overcrowding stress, for the remainder of the 10-day modeling period (Figure 1C4, Figure 2H).
      NOTE: Naïve mice are gently transferred by the tail to a temporary cage, then returned to their home cage without any stress exposure. Provide food and water ad libitum, as with the MISS mice.

4. Behavioral tests

NOTE: Behavioral experiments are conducted within one week following the modeling to assess depressive-like and anxiety-like behaviors in mice. Prior to each test, female mice are moved into the behavioral testing room (15-20 lux lighting) for 1 h of habituation. After each test, mice are returned to their original housing facility.

  1. Sucrose preference test
    NOTE: For details, refer to previous reports36,37,38.
    1. Prepare female subject mice, drinking bottles (50 mL) with ball-point tubes, standard mouse cages with steel-wire tops, sucrose, an electronic scale, a graduated cylinder, a stirring rod, weighing paper, water, a notebook, and a pen.
    2. House all female subject mice (naïve and MISS mice) individually.
    3. Fill the bottles with drinking water (~2/3 of the volume).
      NOTE: Ensure there is no leakage from the bottles before starting the experiment.
    4. Provide each mouse with two water-filled bottles placed side by side for 2 days of habituation.
    5. Prepare 1% sucrose water (1 g of sucrose per 99 mL of water).
    6. At the beginning of the test, replace the water in one bottle with 1% sucrose solution. Record the initial weights of both bottles.
    7. After 12 h, record the bottle weights again and switch the positions of the bottles to avoid positional bias.
    8. After 24 h, record the final weights and calculate sucrose preference as:Sucrose preference (%) = (Sucrose intake / Total fluid intake) × 100
    9. At the end of the test, return the mice to their home cages with regular water bottles.
    10. Clean all bottles and ball-point tubes thoroughly with water.
  2. Tail suspension test
    NOTE: This test32 may be conducted on the same or a separate cohort of mice depending on the experimental design. In this study, it was performed 3 days after the sucrose preference test.
    1. Prepare female subject mice, a tail suspension test box (55 cm high × 60 cm wide × 12 cm deep), adhesive tape, a stopwatch, 75% alcohol, and paper towels.
    2. Set up the video tracking system39. Define an active zone and initiate recording.
    3. Clean the test box with 75% ethanol.
    4. Gently tape the mouse ~2 cm from the end of the tail and suspend it upside down, ensuring its head is ~25 cm from the bottom of the box.
      NOTE: Minimize swinging and avoid unnecessary stress.
    5. Record behavior using the tracking software35 6 min. Use immobility time from the last 4 min for analysis.
      NOTE: Immobility is defined as the absence of movement with the body hanging passively. Exclude data if the mouse falls or climbs up.
    6. Carefully detach the tape and return the mouse to its home cage.
  3. Open-field test
    NOTE: For details, refer to previous reports40,41.
    1. Prepare female subject mice, an open-field test box (40 cm × 40 cm × 40 cm), 75% ethanol, and paper towels.
    2. Set up the video tracking system. Virtually divide the arena into 4 × 4 subareas; define the central 4 units as the central zone, and the remainder as the peripheral zone.
    3. Clean the test box with 75% ethanol.
    4. Place the subject mouse gently in the center of the box, with its head facing the same direction. Allow it to explore for 5 min.
      NOTE: Use a pallet for transfer to reduce handling stress.
    5. Record the total distance traveled and time spent in the central zone using the video tracking system.
    6. At the end of the test, return the mouse to its home cage and clean the apparatus.
  4. Elevated plus maze test
    NOTE: For details, refer to the previous report32.
    1. Prepare female subject mice, an elevated plus maze, 75% ethanol, and paper towels.
      NOTE: The maze consists of two perpendicular runways (50 cm above the floor), each with two arms (30 cm long × 5 cm wide). One set has 15 cm high walls (closed arms), while the other set is open. The central area is 5 cm × 5 cm.
    2. Set up the video tracking system and define the open arms, closed arms, and central area.
    3. Clean the maze with 75% ethanol.
    4. Gently place the mouse in the center of the maze, facing an open arm. Allow free exploration for 5 min.
      NOTE: Use a pallet to minimize stress during placement.
    5. Record time spent in each arm using the tracking system.
      NOTE: If a mouse falls off the maze, exclude its data.
    6. Return the mouse to its home cage and clean the maze thoroughly.

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Results

To investigate whether MISS exposure induces depressive-like behavioral changes, female C57BL/6J mice (n = 16 per group; sample size determined via power analysis, see Supplementary File 1 and Supplementary File 2) subjected to the MISS paradigm were evaluated using the sucrose preference and tail suspension tests, which assess core symptoms of depression-anhedonia and behavioral despair, respectively (Figure 3A-F)....

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Discussion

An increasing number of studies have focused on developing female-specific models for psychiatric disorders using chronic social defeat stress (CSDS) paradigms11,13,14. For example, in two recently established female CSDS models, male aggression toward female mice was artificially induced by either coating female mice with male urine or by chemogenetically activating the ventrolateral subdivision of the ventromedial hypothalamus...

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Disclosures

The authors declare no competing interests.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (31970937 and 82271255), Jiangsu Province Innovative and Entrepreneurial Team Program, Jiangsu Province Key R&D Program Social Development Project (BE2023690), and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX22_2932, KYCX23_2952).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
75% alcoholAladdinA171299-500 mLUsed to sterilize and clean laboratory equipment and instruments
Bottle with rollerball spoutCustom-crafted-Drinking water bottle (50 ml) with rubber stopper with ball spout for the sucrose preference test
C57BL/6J female mice Beijing Vital River Laboratory Animal Technology Co., Ltd219C57BL/6J female mice aged 7-8 weeks are used as subject mice, and mice aged 10-14 weeks are used as winner candidates
C57BL/6J male mice C57BL/6J female mice aged 7-8 weeks are used as subject mice, and mice aged 10-14 weeks are used as winner candidates219C57BL/6J male mice at age of 8-16 weeks as intruders for aggressive CD1 screening.
Elevated plus mazeCustom-crafted-The elevated plus maze consists of perpendiculars, intersecting elevated runways (50 cm above the floor). Each runway has two opposing arms (30 cm long and 5 cm wide). One of the runways has tall walls (15 cm high, closed arms), and the other one has no tall walls (open arms). The intersect area is called the central area (5 cm × 5 cm)
Male CD-1 retired breeder mice Beijing Vital River Laboratory Animal Technology Co., Ltd201Male CD-1 retired breeder mice aged 4-6 months as aggressor candidates
Open-field test boxCustom-crafted-White acrylic open box (L × W × H: 40 cm × 40 cm × 40 cm) for the open-field test
Perforated coverCustom-crafted-Transparent perforated cover (L × W × H: 8 cm × 8 cm × 5.5 cm) for protection of females from direct physical contact with CD1 mice (with 5 × 5 holes in the top ceiling and 3 × 5 holes in the sidewalls, every hole has a 0.5 cm diameter)
SleeveCustom-crafted-Non-transparent plastic sleeve (L × W × H: 14 cm × 10 cm × 20 cm) for holding mice in crowding exposure
Standard mouse cage with steel-wire topSuzhou Fengshi Laboratory Equipment Co., Ltd.FS-SAGIVC-X30Standard mouse cage (L × W × H: 37 cm × 16 cm × 12 cm) with steel-wire top 
SucroseSigmaV900116Used to prepare 1% sucrose solution in the sucrose preference test
Tail suspension boxCustom-crafted-White acrylic box (H × W × D: 55 cm × 60cm × 12cm) for the tail suspension test  
Transparent acrylic tubesCustom-crafted-Transparent acrylic tubes with 26mm inner diameter: 30 mm long for tube competition, 10mm long for tube adaptation.
Video tracking system (SMART V3.0)Panlab76-0695Used to track, record and analyze the activity trajectories and durations of the mice's movements

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Social CompetitionVicarious Social DefeatOvercrowding StressSocial IsolationSucrose Preference TestTail Suspension TestElevated Plus Maze
A Multiple Integrated Social Stress Model for Psychiatric Disorders in Female C57BL/6J Mice
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