This work is supported by the National Institutes of Health (NIH) (Grant No. T32-MH125786 [to EH], Grant Nos. P50-MH115874 and R01-MH108665 [to KJR]), the Eric Dorris Memorial Research Award (to EH), and the McLean Frazier Fund (to KJR).

All experiments were approved by and performed in accordance with McLean Hospital Institutional Animal Care and Use Committee or VA Boston Institutional Animal Care and Use Committee guidelines. All mice were housed in a 12 h light/dark cycle and provided food and water ad libitum. See Figure 1 for a schematic overview of the protocol and the Table of Materials for details about all animals, materials, and equipment used in this protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67113/67113fig01.jpg" /> 
Figure 1: Schematic of experimental design for eaCSDS. (A) Timeline for C57 defeated and control animals. (B) Stages of eaCSDS presented in chronological order, beginning with aggressor screening and followed by repeated social stress for the experimental group, housing for both control and experimental groups, and finally, behavioral testing. Abbreviations: eaCSDS = early adolescent chronic social defeat stress; OFSI = open field social interaction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67113/67113fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Screening aggressive CFW males
<ol>
	<li>Order C57 males such that they arrive at 3 weeks old from an external laboratory to ensure adequate time for aggressors to be screened and prepared for defeats before adolescents reach 4 weeks of age. Leave C57s group housed until 1 week after their arrival. 
	NOTE: Alternatively, animals can be bred inhouse. There have been no observed differences in defeat susceptibility based on whether mice are bred inhouse or shipped from an external laboratory.</li>
	<li>Order adult (8-12 weeks, no breeding experience) CFW males and adult (&#62;8 weeks) CFW ovariectomized (OVX) females. Order as many CFW males as C57s to be defeated, plus at least 50% extra in case aggression decreases over the defeat. For example, if defeating 10 adolescent C57s, order 15 CFW males and 15 CFW OVX females. At least 10 of the CFW males will become aggressive so, if required, use the other 5 non-aggressive males later as stimulus mice during OFSI. 
	NOTE: Nearly 80% of these CFW males will become aggressive after pair-housing.</li>
	<li>One day after their arrival, pair-house CFW males with CFW OVX females (1 male CFW and 1 OVX female CFW per cage) for at least 1 week to allow for sexual experience and increase territorial aggression in the male. 
	NOTE: *CFWs can be pair housed until the defeat begins.</li>
	<li>After 1 week, perform aggression testing:
	<ol>
		<li>Take cages of pair housed CFWs to the testing room.</li>
		<li>Remove the CFW OVX female from the cage and place it in a clean, empty cage.</li>
		<li>Introduce the stimulus C57 male to cage with the CFW male for 1 min.</li>
		<li>Record the latency of attack. If an attack occurs, allow the attack to proceed until 1 min from the introduction of the stimulus C57 has elapsed.</li>
		<li>Remove the stimulus C57, place the CFW OVX female back in the cage with the CFW male, and return them to the housing room.</li>
	</ol>
	</li>
	<li>Repeat this protocol for 2 more days. Use the mice showing an attack latency of &#60;30 s for at least 2 days of screening for adolescent defeats. Continue housing aggressive CFW males with CFW OVX females until the first day of defeat. 
	NOTE: Though there have been no difficulties in eliciting aggression from CFW males after housing with OVX CFW females, removal of the ovaries stops the estrous cycle and can potentially influence sexual behavior, which could result in a lack of sexual experience in CFW males paired with CFW OVX females11,12. The use of females with ovarian ligation rather than ovariectomy is an option that may be used if difficulties in increasing CFW male aggression occur.</li>
	<li>Group house CFW males that either do not attack or show an attack latency of &#62;30 s on 2 or more days. Use these non-aggressive CFW males as stimulus animals during OFSI.</li>
</ol>
2. Handling control adolescent C57 males
<ol>
	<li>When adolescent C57s are 4 weeks old, weigh and record the weight of each animal.</li>
	<li>Separate animals into two equal cohorts (control and defeated), ensuring that the weights between the two cohorts are not significantly different.</li>
	<li>House one control adolescent C57 on one side of a plexiglass partition, with another control adolescent C57 on the other side of the partition (i.e., two control adolescents per cage).</li>
	<li>Store the control cages in a separate room from where the defeats will be taking place.</li>
	<li>For days 1-10, weigh and switch the control adolescent to the opposite cage side every day for 10 days total.</li>
	<li>On the eleventh day, house each control adolescent beside a different control adolescent with a plexiglass partition between the two for the remainder of the experiment. 
	NOTE: This is to ensure both defeat and control animals are housed beside an unfamiliar animal of the same treatment type during the post defeat phase.</li>
</ol>
3. Defeating experimental adolescent C57 males
<ol>
	<li>Weigh and record the weight of adolescent C57s to be defeated.</li>
	<li>Before beginning the defeat, take careful note of the following to maintain the health of the adolescents throughout defeats:
	<ol>
		<li>Count each &#39;compound bite&#39;, or bites in which the resident bites and remains biting, then kicks the stomach of the adolescent, as two separate bites-these types of bites have higher bite force and can break the skin more easily. End the defeat immediately if adolescents receive more than two consecutive compound bites.</li>
		<li>End the defeat immediately if any blood or open wounds become visible. If wounds are observed after the defeat ends, apply antibiotic cream to the wound site. If any wounds larger than 4 mm in diameter occur, euthanize the mouse immediately.</li>
		<li>For adolescents that have open wounds, limit subsequent defeats to no more than five bites until the wound is no longer visible. After the wound has healed, return to the original defeat guidelines of 30 bites or 5 min per defeat.</li>
	</ol>
	</li>
	<li>Remove the CFW OVX female from the cage with the resident CFW male and house it with other OVX females.</li>
	<li>Introduce the adolescent C57 to the cage of the resident CFW male and start the stopwatch.</li>
	<li>Record the aggressor identity, attack latency, number of bites (with clicker counter), and the time it takes for the adolescent to receive 30 bites.</li>
	<li>Remove the adolescent from the cage after 30 bites or 5 min, whichever occurs first.</li>
	<li>Place the plexiglass partition in the middle of the CFW male resident&#39;s cage, with the CFW resident on one side and the defeated adolescent on the other. Ensure that the partition is firmly in place and cannot be moved by the resident.</li>
	<li>In 24 h, repeat the defeat (steps 3.1-3.7) with a different CFW male aggressor.</li>
	<li>Repeat the defeat every 24 h for 10 days total, each day with a different aggressor.</li>
	<li>On the eleventh day, house the defeated adolescents beside the defeated adolescents, with a plexiglass partition between them for the duration of the experiment. Place the defeated adolescents in the same room as controls but on a separate shelf.</li>
	<li>Pair-house the CFW males with any CFW OVX females until the next set of defeats. 
	NOTE: The CFW OVX females used do not need to be the same as those previously paired with the CFW males.</li>
</ol>
4. Testing social avoidance after defeat with open field social interaction testing (OFSI)
<ol>
	<li>Habituate control and defeated animals to the testing room for 1 h.</li>
	<li>Record all sessions with animal tracking software. Place the wire cup against the left wall of the 44 x 44 x 44 cm acrylic arena at the midpoint of the wall and draw a 2 cm circular interaction zone around the cup. 
	NOTE: Ensure that the cup is placed in the same position every time to prevent changes in zones with each trial.</li>
	<li>Place the C57 in the corner of the arena with an empty wire cup for 150 s.</li>
	<li>Remove the C57 from the arena and place it in a clean, empty cage. Place a nonaggressive, unfamiliar CFW adult male inside the wire cup, then place the wire cup inside the arena.</li>
	<li>Reintroduce the C57 to the arena with the CFW-containing wire cup for 150 s.</li>
	<li>Return both animals to their respective cages.</li>
	<li>Clean the arena with water, cleaning any areas with urine or feces with 70% ethanol, before testing the next C57.</li>
	<li>Calculate the length of time any body point is within the interaction zone for the session with the empty cup and the session with the CFW-containing cup. Divide the time spent in the interaction zone with the CFW-containing cup by the time spent in the interaction zone with the empty cup to produce the social interaction ratio.</li>
	<li>Perform the OFSI at any time after the 10 day defeat depending on the developmental window of interest. For eaCSDS, perform OFSI 1 day, 2 weeks, and 5 weeks after the date of the C57&#39;s final defeat.
	<ol>
		<li>A social interaction ratio &#62; 1 is considered typical for control animals. While most control animals will have a social interaction ratio above one, a small subset may have social interaction ratios &#60; 1. For defeated animals, consider those with a social interaction ratio &#60; 1 susceptible and those with a social interaction ratio &#8805; 1 resilient.</li>
	</ol>
	</li>
</ol>

Modeling Early Life Trauma in Mice to Study PTSD and Social Avoidance Behavior

<ol>
	<li>Colich, N. L., Rosen, M. L., Williams, E. S., McLaughlin, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+aging+in+childhood+and+adolescence+following+experiences+of+threat+and+deprivation:+A+systematic+review+and+meta-analysis.">Biological aging in childhood and adolescence following experiences of threat and deprivation: A systematic review and meta-analysis.</a> Psychol Bull. 146 (9), 721-764 (2020).</li><li>Lange, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobehavioural+mechanisms+of+threat+generalization+moderate+the+link+between+childhood+maltreatment+and+psychopathology+in+emerging+adulthood.">Neurobehavioural mechanisms of threat generalization moderate the link between childhood maltreatment and psychopathology in emerging adulthood.</a> J Psychiatry Neurosci. 44 (3), 185-194 (2019).</li><li>McLaughlin, K. A., Sheridan, M. A., Lambert, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+adversity+and+neural+development:+Deprivation+and+threat+as+distinct+dimensions+of+early+experience.">Childhood adversity and neural development: Deprivation and threat as distinct dimensions of early experience.</a> Neurosci Biobehav Rev. 47, 578-591 (2014).</li><li>Keding, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterns+of+delayed+emotion+circuit+maturation+in+abused+girls+with+and+without+internalizing+psychopathology.">Differential patterns of delayed emotion circuit maturation in abused girls with and without internalizing psychopathology.</a> Am J Psychiatry. 178 (11), 1026-1036 (2021).</li><li>Tractenberg, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+maternal+separation+effects+on+behavioural+outcomes+in+mice:+Evidence+from+a+four-stage+methodological+systematic+review.">An overview of maternal separation effects on behavioural outcomes in mice: Evidence from a four-stage methodological systematic review.</a> Neurosci Biobehav Rev. 68, 489-503 (2016).</li><li>Gallo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+bedding+and+nesting+induces+maternal+behavior+resembling+both+hypervigilance+and+abuse.">Limited bedding and nesting induces maternal behavior resembling both hypervigilance and abuse.</a> Front Behav Neurosci. 13, 167 (2019).</li><li>Stevens, J. S., van Rooij, S. J. H., Jovanovic, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+contributors+to+trauma+response:+the+importance+of+sensitive+periods,+early+environment,+and+sex+differences.">Developmental contributors to trauma response: the importance of sensitive periods, early environment, and sex differences.</a> Curr Top Behav Neurosci. 38, 1-22 (2016).</li><li>Berton, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+BDNF+in+the+mesolimbic+dopamine+pathway+in+social+defeat+stress.">Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress.</a> Science (1979). 311 (5762), 864-868 (2006).</li><li>Golden, S. A., Covington, H. E., Berton, O., Russo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+repeated+social+defeat+stress+in+mice.">A standardized protocol for repeated social defeat stress in mice.</a> Nat Protoc. 6 (8), 1183-1191 (2011).</li><li>Kudryavtseva, N. N., Bakshtanovskaya, I. V., Koryakina, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+model+of+depression+in+mice+of+C57BL/6J+strain.">Social model of depression in mice of C57BL/6J strain.</a> Pharmacol Biochem Behav. 38 (2), 315-320 (1991).</li><li>Huijgens, P. T., Guarraci, F. A., Olivier, J. D. A., Snoeren, E. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+rat+sexual+behavior:+Insights+from+inter-copulatory+intervals.">Male rat sexual behavior: Insights from inter-copulatory intervals.</a> Behav Processes. 190, 104458 (2021).</li><li>Zucon Bacelar, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aged+and+induced-premature+ovarian+failure+mouse+models+affect+diestrus+profile+and+ovarian+features.">Aged and induced-premature ovarian failure mouse models affect diestrus profile and ovarian features.</a> PLoS One. 18 (12), e0284887 (2023).</li><li>Mouri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+adolescent+social+defeat+stress+exposure+persistently+impairs+social+behaviors+and+neurogenesis.">Early adolescent social defeat stress exposure persistently impairs social behaviors and neurogenesis.</a> Neuropharmacology. 133, 23-37 (2018).</li><li>Hasegawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+serotonergic+and+dopaminergic+neuronal+systems+in+the+antidepressant-resistant+impairment+of+social+behaviors+induced+by+social+defeat+stress+exposure+as+early+adolescents.">Dysfunction of serotonergic and dopaminergic neuronal systems in the antidepressant-resistant impairment of social behaviors induced by social defeat stress exposure as early adolescents.</a> Int J Neuropsychopharmacol. 21 (9), 837-846 (2018).</li><li>Pantoja-Urb&#225;n, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gains+and+losses:+resilience+to+social+defeat+stress+in+adolescent+female+mice.">Gains and losses: resilience to social defeat stress in adolescent female mice.</a> Biol Psychiatry. 95 (1), 37-47 (2023).</li><li>Demaestri, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+scarcity+but+not+maternal+separation+provokes+unpredictable+maternal+care+sequences+in+mice+and+both+upregulate+Crh-associated+gene+expression+in+the+amygdala.">Resource scarcity but not maternal separation provokes unpredictable maternal care sequences in mice and both upregulate Crh-associated gene expression in the amygdala.</a> Neurobiol Stress. 20, 100484 (2022).</li><li>Walker, C. -. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+early+life+stress+induced+by+limited+bedding+and+nesting+(LBN)+material+in+rodents:+critical+considerations+of+methodology,+outcomes+and+translational+potential.">Chronic early life stress induced by limited bedding and nesting (LBN) material in rodents: critical considerations of methodology, outcomes and translational potential.</a> Stress. 20 (5), 421-448 (2017).</li><li>Pardo, G. V. E., Alfaro Saca, E. E., Becerra Flores, C. T., Delgado Cas&#243;s, W. F., Pacheco-Otalora, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+bedding+nesting+paradigm+alters+maternal+behavior+and+pup&#8217;s+early+developmental+milestones+but+did+not+induce+anxiety-+or+depressive-like+behavior+in+two+different+inbred+mice.">Limited bedding nesting paradigm alters maternal behavior and pup&#8217;s early developmental milestones but did not induce anxiety- or depressive-like behavior in two different inbred mice.</a> Dev Psychobiol. 65 (1), e22357 (2023).</li><li>Raineki, C., Cort&#233;s, M. R., Belnoue, L., Sullivan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+early-life+abuse+differ+across+development:+infant+social+behavior+deficits+are+followed+by+adolescent+depressive-like+behaviors+mediated+by+the+amygdala.">Effects of early-life abuse differ across development: infant social behavior deficits are followed by adolescent depressive-like behaviors mediated by the amygdala.</a> J Neurosci. 32 (22), 7758-7765 (2012).</li><li>Rinc&#243;n-Cort&#233;s, M., Sullivan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+social+behavior+deficit,+blunted+corticolimbic+activity+and+adult+depression-like+behavior+in+a+rodent+model+of+maternal+maltreatment.">Emergence of social behavior deficit, blunted corticolimbic activity and adult depression-like behavior in a rodent model of maternal maltreatment.</a> Transl Psychiatry. 6 (10), e930 (2016).</li><li>Raineki, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+neurobehavioral+rescue+by+memories+of+early-life+abuse:+the+safety+signal+value+of+odors+learned+during+abusive+attachment.">Paradoxical neurobehavioral rescue by memories of early-life abuse: the safety signal value of odors learned during abusive attachment.</a> Neuropsychopharmacology. 40 (4), 906-914 (2015).</li><li>Niwa, M., Matsumoto, Y., Mouri, A., Ozaki, N., Nabeshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerability+in+early+life+to+changes+in+the+rearing+environment+plays+a+crucial+role+in+the+aetiopathology+of+psychiatric+disorders.">Vulnerability in early life to changes in the rearing environment plays a crucial role in the aetiopathology of psychiatric disorders.</a> Int J Neuropsychopharmacol. 14 (4), 459-477 (2011).</li><li>Franklin, T. B., Linder, N., Russig, H., Th&#246;ny, B., Mansuy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+early+stress+on+social+abilities+and+serotonergic+functions+across+generations+in+mice.">Influence of early stress on social abilities and serotonergic functions across generations in mice.</a> PLoS One. 6 (7), e21842 (2011).</li><li>Ronquillo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+nurture:+Comparing+mouse+behavior+in+classic+versus+revised+anxiety-like+and+social+behavioral+assays+in+genetically+or+environmentally+defined+groups.">Nature and nurture: Comparing mouse behavior in classic versus revised anxiety-like and social behavioral assays in genetically or environmentally defined groups.</a> Genes Brain Behav. 22 (6), e12869 (2023).</li></ol>

KJR has performed scientific consultation for Bioxcel, Bionomics, Acer, Takeda, and Jazz Pharma; serves on Scientific Advisory Boards for Sage and the Brain Research Foundation; has received sponsored research support from Takeda, Brainsway, and Alto Neuroscience; and receives research funding from the NIH. All other authors report no biomedical financial interests or potential conflicts of interest.

Early adolescent chronic social defeat stress (eaCSDS) results in robust social avoidance, as tested in open field social interaction (OFSI) tests, across the lifespan. This protocol was based on a traditional 10 day adult chronic social defeat stress model9 with several small, but key, modifications that differentiate it from traditional adult defeat and other early adolescent defeat protocols13,14,15. The first is the use of younger CFW males that have no breeding experience and are housed with females prior to defeat to increase territorial aggression. One possible explanation for the difficulty producing aggression towards adolescents in other protocols may be the use of retired breeders, which are typically much older and display lower levels of aggression than younger males. The second modification is the use of smaller cages in which mice are defeated and housed, which provides a smaller environment from which the intruder can avoid the resident both during the defeat and during housing after defeat. Finally, the number of bites is quantified to ensure that all mice receive consistent defeat experience.
This model is limited to late juvenile-early adolescent male mice. In its current form, this protocol has only tested defeats beginning at P26 at the youngest. It will be interesting in the future to examine the effects of this protocol on mice immediately after weaning, though the duration and number of bites during attacks may need to be limited given the smaller size of the mice. Notably, this protocol is for use in males, though preliminary evidence indicates it may also prove reliable for early adolescent female defeats with the modification of using female CFWs as aggressors.
Other models of early life stress typically target preweaning windows. Two of these models, maternal separation (MS) and limited bedding and nesting (LBN), are commonly used to induce early life stress before P10. Both MS and LBN affect cortisol levels and expression of corticotropin-releasing hormone (Crh)-related genes in pups16, but their effects on anxiety-like behavior are mixed17,18, and measures of adult social interaction in pups exposed to either paradigm are limited. LBN has been shown to reduce social interaction in adolescence19,20 and adulthood21 in rats, but, to our knowledge, its effects on social interaction in mice have not been reported. MS in C57/B6 mice has mixed effects on social interaction, with some groups reporting reduced social interaction22,23, while a more recent study reports an increase in social interaction24. In contrast to LBN and MS, this model of early adolescent defeat focuses on social interaction as a primary behavioral readout and targets stress to a later development period, which may account for the differences in social interaction across this model and MS and LBN models.
Early life physical abuse is incredibly prevalent in our society and often results in debilitating psychiatric conditions in adulthood. Despite its prevalence, the effects of early life abuse on adult neural circuits are not well understood, in part due to the lack of models of early adolescent physical abuse. The intention behind creating a protocol that facilitates consistent and robust social defeat in early adolescent males is to facilitate continued studies of the neural changes that arise from early life trauma.

As many as 1 in 7 children experience childhood abuse. Along with greatly increasing the risk for development of psychiatric diseases such as PTSD and other fear-related disorders in adulthood, this early life experience has a profound impact on brain anatomy and connectivity1,2. Early life physical abuse has recently been found to delay circuit maturation while early life neglect has found to accelerate it3,4, necessitating the development of models of early life physical trauma in addition to those of neglect such as maternal separation (MS)5 and limited bedding and nesting (LBN)6. Additionally, MS and LBN models are carried out in infant animals before weaning. As trauma during middle childhood (ages 10-12) is especially linked to susceptibility to PTSD and psychiatric disorders later in life7, the ability to model early life stress at developmental stages outside of infancy is critical.
To develop a model of early life trauma with behavioral effects that last into adulthood, this 10 day early adolescent chronic social defeat stress (eaCSDS) protocol was designed with modifications of a traditional adult chronic social defeat stress model8,9,10. The traditional protocol has been unsuccessful in maintaining the long-term aggression towards adolescents that is needed to successfully perform 10 consecutive days of defeat. In this protocol, early adolescent C57/B6J males (4 weeks old, ~P30) are repeatedly defeated by a young (8-10 weeks) adult CFW male that has no breeding experience, a modification of the traditional use of retired CFW male breeders. Another key modification of the traditional protocol is that the aggression of the CFW males is heightened by 1 week of co-housing with an ovariectomized female. After this housing, CFW males rapidly and consistently attack adolescent males, negating the need for stimulus adult C57/B6J mice to increase aggression before defeating adolescent males. Another modification of the traditional adult model is the use of smaller cages (29 cm x 19 cm x 13 cm) during defeats and overnight housing.
Testing of susceptibility to defeat is performed using the open field social interaction test (OFSI). Mice that are susceptible to defeat are classified as animals that spend more time interacting with an empty cup than with a cup with a CFW male inside. The protocol described below produces greater than 60% susceptibility during testing in adolescence and adulthood.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>Quantity (for a cohort of 10 defeated and 10 control animals)</td>
		</tr>
		<tr>
			<td>Arena, opaque</td>
			<td>generic</td>
			<td>---</td>
			<td>1 
			Size: 44 x 44 x 44 cm</td>
		</tr>
		<tr>
			<td>CFW male (8-10 weeks)</td>
			<td>Charles River</td>
			<td>24</td>
			<td>15</td>
		</tr>
		<tr>
			<td>CFW OVX female (8+ weeks)</td>
			<td>Charles River</td>
			<td>24</td>
			<td>15</td>
		</tr>
		<tr>
			<td>C57/B6J male (3 weeks)</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td>20</td>
		</tr>
		<tr>
			<td>C57/B6J male (6-7 weeks, stimulus mice)</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Clicker counter</td>
			<td>generic</td>
			<td>---</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Home cages for housing during defeats</td>
			<td>generic</td>
			<td>---</td>
			<td>15 (10 for defeated animals, 5 for control animals) 
			Size: 29 x 19 x 13 cm</td>
		</tr>
		<tr>
			<td>Plexiglass partitions</td>
			<td>generic</td>
			<td>---</td>
			<td>15 
			Size: To fit 29 x 19 x 13 cm mouse cage</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>generic</td>
			<td>---</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>generic</td>
			<td>---</td>
			<td>1 
			Size: &#62;15 frames per second capability</td>
		</tr>
		<tr>
			<td>Wire cup</td>
			<td>generic</td>
			<td>---</td>
			<td>1 
			Size: 10 cm diameter, 15 cm height</td>
		</tr>
	</tbody>
</table>

chronic social defeat stress in early adolescent male mice

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, such as Post-Traumatic Stress Disorder (PTSD), in adolescence and adulthood. Hallmark behavioral symptoms of PTSD include avoidance of cues and contexts associated with trauma. The ability to model long-lasting changes in the brain due to early life trauma is critical to determine potential circuit and molecular targets for the modulation of resultant symptoms. This manuscript describes a protocol for modeling early life physical and psychological trauma in early adolescent male mice that produces socially avoidant behavior. Adolescent male mice are exposed to repeated aggressive encounters followed by overnight housing, which provides an added dimension of psychological stress, with an adult male aggressor every day for 10 days. Repeated social defeat paired with overnight housing by the aggressor in early adolescence produces robust social avoidance behavior that lasts throughout adulthood. Social avoidance can be readily quantified in early adolescents, adolescents, and adults using open field social interaction testing. Early adolescent social defeat robustly produces more than 60% susceptible animals in adulthood.

eaCSDS produces robust reductions in social interaction in OFSI across a large number of animals (n = 50 animals) (Figure 2A-C); defeated animals show decreased measures of both social interaction ratio and raw social interaction time in comparison to controls at all time points tested. When tested 1 day after defeats, adolescent (~P40) defeated mice show a significant decrease in social interaction ratio and raw social interaction time from controls but no significant difference in raw time spent with an empty cup (control: 65.48 &#177; 4.99 s, defeat: 67.75 &#177; 3.86 s; two-tailed unpaired t-test, p = 0.72). This decrease in social interaction and raw social interaction time is greatest when animals are tested 2 weeks after defeat in late adolescence (~P54). Interestingly, at this time point, defeated mice show a significant increase in the raw time spent with an empty cup in addition to the significant decreases in social interaction score and raw social interaction time (controls: 51.58 &#177; 3.53 s, defeated: 68.62 &#177; 2.86 s; two-tailed unpaired t-test, p = 0.0003).
The decrease in social interaction and raw social interaction time when tested 5 weeks after defeat remains robust even in adulthood (~P70), but as seen 24 h after defeat, there is no significant difference in raw time spent with an empty cup (52.83 &#177; 10.13, 53.15 &#177; 3.48; two-tailed unpaired t-test, p = 0.97). Within a single cohort, there can be variability in the proportion of susceptibility. However, cohorts tested using this protocol have consistently produced susceptibility in adulthood in 60% or greater of defeated animals. A cohort with a higher percentage of susceptible animals (90%) in adulthood (Figure 2D-F) versus a cohort with a lower percentage of susceptible animals (71%) (Figure 2G-I) in adulthood is shown for reference. Summed heatmaps from the social interaction portion of OFSI are shown for 10 control and 10 defeated animals at 24 h, 2 weeks, and 5 weeks post defeat (Figure 3) to provide visual examples of how defeated mice consistently spend an increased amount of time in corners of the arena and a decreased amount of time inside the interaction zone.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67113/67113fig02.jpg" /> 
Figure 2: eaCSDS produces robust social avoidance in OFSI. (A-C) SI scores (left) and raw social interaction times (right) across 46 control and 50 defeat males (A) 1 day (~P40), (B) 2 weeks (~P54), and (C) 5 weeks (~P70) after defeat. (D-F) SI scores and raw social interaction times for an example cohort with a high percentage (90%) of susceptible animals in adulthood (as determined by average SI score of defeated adults) (n = 10 control and 10 defeated males) (D) 1 day, (E) 2 weeks, and (F) 5 weeks after defeat. (G-I) SI scores and raw social interaction times for an example cohort with a lower percentage (71%) of susceptible animals in adulthood (as determined by average SI score of defeated adults) (n = 10 control and 14 defeated males) (G) 1 day, (H) 2 weeks, and (I) 5 weeks after defeat. All comparisons were performed with unpaired two-tailed t-tests. **, p &#60; 0.01; ***, p &#60; 0.001; ****, p &#60; 0.0001. Abbreviations: eaCSDS = early adolescent chronic social defeat stress; OFSI = open field social interaction; SI = social interaction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67113/67113fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67113/67113fig03.jpg" /> 
Figure 3: OFSI Heatmaps during social interaction in control and defeated animals. Heatmaps from the social interaction portion of OFSI testing (i.e., when nonaggressive CFW male is inside the cup) 24 h, 2 weeks, and 5 weeks after defeat. Note that each heatmap is summed over 10 animals (controls: n = 10 animals, grey; defeated: n = 10 animals, purple). Blue male symbol indicates location of cup with nonaggressive CFW male inside. White circle indicates location of interaction zone. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67113/67113fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Chronic Social Defeat Stress in Early Adolescent Male Mice

This protocol details how to perform 10 day chronic social defeat stress in early adolescent male mice to produce long-lasting social avoidance in adulthood in more than 60% of mice.

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, ...

chronic-social-defeat-stress-in-early-adolescent-male-mice

Nanyang Technological University

Research

JoVE Journal

Behavior

284 Views.  VA Boston. This protocol details how to perform 10 day chronic social defeat stress in early adolescent male mice to produce long-lasting social avoidance in adulthood in more than 60% of mice.

Video: Chronic Social Defeat Stress in Early Adolescent Male Mice

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

A Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...