The authors wish to acknowledge the Netherlands Organization for Scientific Research (NWO) for their generous support to publish this manuscript as an open access paper.

1.	The Experimental Setup
<ol>
 <li>Use for each resident a cage with a floor space of about half a square meter. Offensive aggression is a highly active form of behavior that requires sufficient space for its full expression. The cage should be made of sanitizable material. </li>
 <li>House each resident with a female for at least one week before the start of the experiments, which will facilitate the development of territoriality. At the same time this will prevent social isolation, which is known to be stressful for social animals and may lead to reduced welfare and aberrant forms of social behavior. </li>
 <li>Use companion females that are sterilized by ligation of the oviducts. In this way, the female stays hormonally intact and will be regularly receptive without becoming pregnant and developing maternal aggression. </li>
</ol>
2.	Procedure
<ol>
 <li>House the resident male and the companion female together in the resident cage for at least one week prior to testing. </li>
 <li>Do not clean the bedding of the cage during that initial week or prior to later testing, since territoriality is strongly based on the presence of olfactory cues. These cues are both important for the resident in establishing its own territory and for the intruder to know that it is in the home cage of the resident. Please, notice that this deviation from the general animal care taking procedures may require special permission from the authorities. </li>
 <li>Remove the companion female from the residential cage one hour before the test. </li>
 <li>Introduce an unfamiliar male into the home cage of the resident at the start of the test. Preferably, the intruder should be slightly smaller than the resident and should not have been used in previous interactions with the same resident. </li>
 <li>Record the behavior of the resident, preferably using a light sensitive video camera. </li>
 <li>A test duration of 10 min is usually sufficient for the expression of the full offensive behavioral repertoire. For the purpose of standardization one may consider to continue recording for ten minutes after the first attack. </li>
 <li>After completion of the test, remove the intruder male from the cage and reunite the resident male with its companion female. </li>
 <li>Although aggression may occur at all times of day, it is best to test only during the dark phase; the rats' main activity phase. </li>
 <li>Testing can be performed once or twice per day. The level of aggressive behavior often increases across the first couple of tests but generally stabilizes after three to four tests. </li>
</ol>
3.	Offensive Aggressive Behavior
<ol>
 <li>In principle, any strain of rats can be used as residents. However, strains may differ considerably for their absolute level of aggressive behavior. Moreover, there may be a considerable individual variation within strains. </li>
 <li>Standardize intruders as much as possible in terms of strain, age and weight. Use rats of a non-aggressive strain that are slightly lower in body weight than the resident male. </li>
 <li>Determine in the resident male the duration and frequency of the following behavioral parameters: 
 <ol class="lroman">
 <li>Attack latency: the time between the introduction of the intruder and the first clinch attack</li>
 <li>Move towards</li>
 <li>Social exploration </li>
 <li>Ano-genital sniffing </li>
 <li>Rearing </li>
 <li>Lateral threat </li>
 <li>Upright posture </li>
 <li>Clinch attack </li>
 <li>Keep down </li>
 <li>Chase </li>
 <li>Non-social explore </li>
 <li>Rest or inactivity </li>
 </ol>
 </li>
 <li>Analyses 
 <ol class="lroman">
 <li>The behavioral parameters recorded should cover 100% of the observation time. This facilitates an unbiased interpretation of the results, i.e. when one behavior goes up, another has to go down. </li>
 <li>Data reduction can be obtained by calculating scores of different behavioral categories; particularly:
 <ol>
 <li>Total offense score: sum of lateral threat, upright, clinch, keep down and chase</li>
 <li> 	Social exploration score: sum of social explore, ano-genital sniffing and social groom </li>
 </ol>
 </li>
 <li>Data can be expressed as percentages of the total observation time </li>
 </ol>
 </li>
</ol>
4.	Violence
<ol>
 <li>Follow the protocol explained under section 2 </li>
 <li>Behavioral tests and criteria: 
 <ol class="lroman">
 <li>Determine in the resident male the attack latency. A very short latency is a first indicator of violence.</li>
 <li>Calculate for the resident male the ratio between the frequencies of lateral threat and clinch. A ratio below 1 shows that animals attack without any introductory behavior and forewarning which is a reliable indicator of violence (out of control). </li>
 <li>Bite target sites: 
 <ol>
 <li>Bites attacks targeted at vulnerable body parts such as the throat, belly and paws are a sign of violence. </li>
 <li>Bites targeted at the snout of the opponent indicate defensive behavior of the actor.</li>
 </ol>
 </li>
 <li>Out of context tests: 
 <ol>
 <li>Use an unfamiliar female as intruder. Attack of a female is a sign of violence.</li>
 <li>Use an anesthetized male as intruder. Attack of an anesthetized male is a sign of violence.</li>
 <li>Test the aggressive behavior of the resident in a novel environment. The absence of a change in aggression compared to the home cage is a sign of violence.
 The endpoint of this test is the first attack or ten minutes when the resident does not attack. </li>
 </ol>
 </li>
 </ol>
 </li>
 </ol>
5.	Defensive Behavior 
<ol>
 <li>Select a number of well trained, highly aggressive, non-violent resident males </li>
 <li>Follow the protocol as explained under section 2 </li>
 <li>Determine in the intruder the duration and frequency of the following behavioral parameters (in addition to 3c): 
 <ol class="lroman">
 <li>Submission latency </li>
 <li>Submissive posture </li>
 <li>Move away </li>
 <li>Flight </li>
 <li>Defensive upright posture</li>
 <li>Freeze </li>
 <li>Non-social explore </li>
 <li>Rearing </li>
 </ol>
 </li>
 <li>Analysis 
 <ol class="lroman">
 <li>Data reduction can be obtained by calculating a defense score, which is the sum of the amount of time spent on flight, defensive upright posture, submission and freeze </li>
 </ol>
 </li>
</ol>
6.	Social Stress
<ol>
 <li>Follow the protocol as explained under section 5</li>
 <li>For intermittent social stress, repeat the protocol </li>
 <li>For chronic social stress, keep the intruder in the resident's cage, but separated from the resident by a wire mesh screen
 </li>
 <li>Continue step b and/or c for as long as the scientific questions and hypotheses require.</li>
</ol>

<ol>
	<li>Albert, D. J., Dyson, E. M., Walsh, M. L., Petrovic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohabitation+with+a+female+activates+testosterone-dependent+social+aggression+in+male+rats+independently+of+changes+in+serum+testosterone+concentration.">Cohabitation with a female activates testosterone-dependent social aggression in male rats independently of changes in serum testosterone concentration.</a> Physiology &amp; Behavior. 44 (6), 735-740 (1988).</li><li>Barnett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+social+behaviour+in+wild+rats.">An analysis of social behaviour in wild rats.</a> Proceedings of the Zoological Society London. 130, 107 (1958).</li><li>Berton, O., McClung, C. A., Dileone, R. J., Krishnan, V., Renthal, W., Russo, S. 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=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. (New York, N.Y.). 311 (5762), 864-868 (2006).</li><li>Blanchard, R. J., Blanchard, D. C., Takahashi, T., Kelley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attack+and+defensive+behaviour+in+the+albino+rat.">Attack and defensive behaviour in the albino rat.</a> Animal Behaviour. 25 (3), 622-634 (1977).</li><li>Blanchard, R. J., Wall, P. M., Blanchard, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Problems+in+the+study+of+rodent+aggression.">Problems in the study of rodent aggression.</a> Hormones and Behavior. 44 (3), 161-170 (2003).</li><li>Bouwknecht, J. A., Hijzen, T. H., van der Gugten, J., Maes, R. A., Hen, R., Olivier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+5-HT(1B)+receptors+is+associated+with+impaired+impulse+control+in+male+5-HT(1B)+knockout+mice.">Absence of 5-HT(1B) receptors is associated with impaired impulse control in male 5-HT(1B) knockout mice.</a> Biological Psychiatry. 49 (7), 557-568 (2001).</li><li>de Boer, S. F., Caramaschi, D., Natarajan, D., Koolhaas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vicious+cycle+towards+violence:+Focus+on+the+negative+feedback+mechanisms+of+brain+serotonin+neurotransmission.">The vicious cycle towards violence: Focus on the negative feedback mechanisms of brain serotonin neurotransmission.</a> Frontiers in Behavioral Neuroscience. 3, 52 (2009).</li><li>de Boer, S. F., Koolhaas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT1A+and+5-HT1B+receptor+agonists+and+aggression:+A+pharmacological+challenge+of+the+serotonin+deficiency+hypothesis.">5-HT1A and 5-HT1B receptor agonists and aggression: A pharmacological challenge of the serotonin deficiency hypothesis.</a> European Journal of Pharmacology. 526 (1-3), 125-139 (2005).</li><li>de Boer, S. F., Van der Vegt, B. J., Koolhaas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+in+aggression+of+feral+rodent+strains:+A+standard+for+the+genetics+of+aggression+and+violence?.">Individual variation in aggression of feral rodent strains: A standard for the genetics of aggression and violence?.</a> Behav. Genet. 33, 485-501 (2003).</li><li>Fish, E. W., DeBold, J. F., Miczek, 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=Escalated+aggression+as+a+reward:+Corticosterone+and+GABA(A)+receptor+positive+modulators+in+mice.">Escalated aggression as a reward: Corticosterone and GABA(A) receptor positive modulators in mice.</a> Psychopharmacology (Berl). 182, 116-127 (2005).</li><li>Grant, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+social+behaviour+of+the+male+laboratory+rat.">An analysis of the social behaviour of the male laboratory rat.</a> Behaviour. 21, 260 (1963).</li><li>Haller, J., Halasz, J., Mikics, E., Kruk, M. R., Makara, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultradian+corticosterone+rhythm+and+the+propensity+to+behave+aggressively+in+male+rats.">Ultradian corticosterone rhythm and the propensity to behave aggressively in male rats.</a> J .Neuroendocrinol. 12, 937-940 (2000).</li><li>Haller, J., Kruk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+and+abnormal+aggression:+Human+disorders+and+novel+laboratory+models.">Normal and abnormal aggression: Human disorders and novel laboratory models.</a> Neuroscience and Biobehavioral Reviews. 30 (3), 292-303 (2006).</li><li>Koolhaas, J. M., Bartolomucci, A., Buwalda, B., de Boer, S. F., Flugge, G., Korte, S. 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=Stress+revisited:+A+critical+evaluation+of+the+stress+concept.">Stress revisited: A critical evaluation of the stress concept.</a> Neuroscience and Biobehavioral Reviews. 35 (5), 1291-1301 (2011).</li><li>Koolhaas, J. M., De Boer, S. F., Coppens, C. M., Buwalda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroendocrinology+of+coping+styles:+Towards+understanding+the+biology+of+individual+variation.">Neuroendocrinology of coping styles: Towards understanding the biology of individual variation.</a> Front Neuroendocrinol. , (2010).</li><li>Koolhaas, J. M., Meerlo, P., De Boer, S. F., Strubbe, J. H., Bohus, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+temporal+dynamics+of+the+stress+response.">The temporal dynamics of the stress response.</a> Neuroscience and Biobehavioral Reviews. 21 (6), 775-782 (1997).</li><li>Koolhaas, J. M., Schuurman, T., Wiepkema, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+intraspecific+agonistic+behaviour+in+the+rat.">The organization of intraspecific agonistic behaviour in the rat.</a> Progress in Neurobiology. 15 (3), 247-268 (1980).</li><li>Krug, E. G., Dahlberg, L. L., Mercy, J. A., Zwi, A. B., Lozano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World report on violence and health. , (2002).</li><li>Litvin, Y., Blanchard, D. C., Pentkowski, N. S., Blanchard, R. 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+pinch+or+a+lesion:+A+reconceptualization+of+biting+consequences+in+mice.">A pinch or a lesion: A reconceptualization of biting consequences in mice.</a> Aggressive Behavior. 33 (6), 545-551 (2007).</li><li>Pijlman, F. T., Wolterink, G., Van Ree, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+emotional+stress+have+differential+effects+on+preference+for+saccharine+and+open+field+behaviour+in+rats.">Physical and emotional stress have differential effects on preference for saccharine and open field behaviour in rats.</a> Behavioural Brain Research. 139 (1-2), 131-138 (2003).</li><li>Popova, N. K., Kulikov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+"spontaneous"+intermale+aggression+in+mice.">Genetic analysis of "spontaneous" intermale aggression in mice.</a> Aggressive Behavior. 12, 425 (1986).</li><li>Ruis, M. A., te Brake, J. H., Buwalda, B., de Boer, S. F., Meerlo, P., Korte, S. 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=Housing+familiar+male+wildtype+rats+together+reduces+the+long-term+adverse+behavioural+and+physiological+effects+of+social+defeat.">Housing familiar male wildtype rats together reduces the long-term adverse behavioural and physiological effects of social defeat.</a> Psychoneuroendocrinology. 24, 285-300 (1999).</li><li>Timmermans, P. J. 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+behaviour+in+the+rat.">Social behaviour in the rat.</a> Unpublished. , (1978).</li></ol>

The authors declare that they have no competing financial interests.

The resident-intruder paradigm can be used to study offensive aggression, defensive behavior, violence and social stress in rats and, with some small modifications for other rodent species as well. When studying aggression, principally all rat strains can be used. However, strains are not equally suitable. Depending on the exact purpose of the experiment, some specific characteristics of the animals should be considered. It is important to notice that there are large strain differences in the level and intensity of offensive aggression shown as a resident. Figure 1 shows the frequency distribution of offensive aggression in an originally feral rat strain (Figure 1a) and in a standard laboratory Wistar strain (Figure 1b). The two strains differ considerably in the number of animals that will show aggressive behavior at all. Moreover, there is a large difference in the absolute scale of offense. The feral rat strain ranges from zero up to 80% offense in our standard 10 min test whereas the Wistar strain has a maximum of 25% offense; the highly aggressive phenotype is absent in this latter strain 16. 
When the resident-intruder paradigm is used to study defensive behavior and social stress in the intruder, one needs these highly aggressive phenotypes as residents. After all, the resident has to reliably defeat any intruder entering his territory. Of course, the most aggressive individuals should be chosen and one should realize that even in a highly aggressive strain, not all individuals will be suitable for this purpose. True adult males of at least four months of age 10 should be used and one may consider using former breeder males. It is recommended to give the resident males some further experience with intruders during the days before the actual start of the social stress experiment. Any strain can be used as intruder. However, to guarantee clear winning by the resident and defeat of the intruder, we advise to use intruders with a slightly lower body weight than the resident male. Because olfactory cues are important in social communication and territoriality, cleaning the cage of the resident prior to testing will be a serious confounder.
	It is recommended to videotape and record the full behavioral repertoire of the experimental animal during the test. This allows an unbiased analysis of the results, i.e. when one behavior goes up, another is likely to go down. For example, the results depicted in Figure 3 shows that a reduction in offense following drug treatment is accompanied by an increase in social exploration and not by immobility. This provides evidence that the drug induced reduction in offense is not due to some kind of a sedative or motor inactivity effect. The total offense score is an index of the intensity of aggression and the latency of the first attack and number of attacking animals can be used as a measure of the readiness to attack 22.
	For social stress experiments one should have a clear criterion for social defeat. When the intruder adopts a submissive posture (see above) and stays in this posture even when the resident moves away, this is a reliable criterion for social defeat. Notice that housing conditions of the intruders are extremely important in social stress research. First, the social stress should not be administered in the same room where the non-stressed controls are housed. Control animals witnessing (social) stress in other individuals may experience major stress themselves 7, 21. Second, the consequences of social stress are sensitive to social buffering effects. Group housed, socially stressed animals do not show the same long-term consequences seen in socially stressed animals that are socially isolated afterwards 23.
	Taken together, the resident intruder paradigm allows research on both the causes and the consequences of aggressive behavior. It is a model with a high face and construct validity that covers not only the adaptive biology of social behavior, but can be used to study maladaptive aspects as well in terms of violence and social stress pathology.

Aggressive behavior belongs to the natural behavioral repertoire of virtually all animal species. From a biological point of view, aggressive behavior can be considered as a highly functional form of social communication aimed at active control of the social environment. It is characterized by a set of species-typical behaviors performed in close interaction with the opponent. Overt aggression and physical conflicts are potentially harmful not only for the victim but for the aggressor as well. Therefore, throughout the animal kingdom, mechanisms have been developed to minimize and control physical aggression in order to prevent its potentially adverse consequences. Such mechanisms include, for example, threatening behavior that often predicts and may thereby prevent physical attacks. Other mechanisms to keep aggression in control are taboos, ritualization, submission, reconciliation and appeasement. This holds in particular for offensive aggression, which is a form of aggressive behavior characterized by initiative of the offender and a range of introductory, often threatening, behavioral displays before attempting to reach the back and neck as non-vulnerable targets for the consummatory aggressive attack bites. Despite such highly adaptive control mechanism, examples exist of functional aggression turning into violence, which can thus be defined as an injurious form of offensive aggression that is out of control and out of context; it is a pathological form of offensive behavior that is no longer subject to inhibitory control mechanisms and that has no additive functional value to normal aggressive behavior in social communication 8. Violence thus differs both quantitatively and qualitatively from normal adaptive offensiveness. This may include bites attacks targeted at vulnerable body parts such as the throat, belly and paws are normally off limits 5, 13, 20, 24.
Defensive aggression is the form of aggressive behavior performed in response to an attack by another individual. It is distinctly different from offense in terms of its behavioral expression and inhibitory controls 5. Note that extreme forms of defensive behavior can have violent characteristics.
Much of the preclinical aggression research is conducted in territorial male resident rats or mice confronting an intruder conspecific. This so-called resident-intruder paradigm allows the spontaneous and natural expression of both offensive aggression and defensive behavior in laboratory rodents in a semi natural laboratory setting. By recording the frequencies, durations, latencies and temporal and sequential patterns of all the observed behavioral acts and postures in the combatants during these confrontations, a detailed quantitative picture (ethogram) of offensive (resident) and defensive (intruder) aggression is obtained. For extensive descriptions of the various behaviors see 3, 12, 18. The paradigm is based on the fact that an adult male rat will establish a territory when given sufficient living space. Territoriality is strongly enhanced in the presence of females and/or sexual experience 1. As a consequence of territoriality, the resident will attack unfamiliar males intruding in its home cage. Hence, offensive aggression can be studied by using the resident as the experimental animal. To determine the violent nature of aggression one can assess whether the offense is out of context and inhibitory control by using different types of intruders such as females or anesthetized males or a novel environment. A detailed quantitative analysis of the offensive behavioral repertoire is required to reveal to what extent the observed aggression is out of control.
The intruders in the resident-intruder paradigm will show defensive behavior in response to the offensive attacks by the resident. The paradigm therefore also allows one to study defensive behavior and social stress by using the intruder as the experimental animal. A form of chronic social stress can be created by repeatedly using the experimental animal as intruder, or by housing it in the cage (territory) of the resident, separated by a wire mesh screen 4.
	Like any kind of stress paradigm, the resident-intruder paradigm is not free from ethical concerns. We therefore want to present a number of ethical considerations. Aggression, violence and social stress are serious problems in our human society. A report of the World Health Organization shows that interpersonal violence is not only a major source of death worldwide it is also a major source of serious health problems in the surviving victims of aggression 19. Hence, there is a need to understand these behaviors in terms of their underlying causal mechanisms and modulating factors. Animal models are essential to obtain experimental support of the causal nature of physiological and environmental factors. From a biological point of view, aggression is a natural, biologically functional form of social behavior aimed at the establishment of a territory, social dominance and defense of resources. The resident-intruder paradigm brings this natural form of behavior into a laboratory setting that allows for controlled studies of both the aggressor and the victim. An issue of ethical concern is the question to what extent the animal's welfare is compromised when exposed to this paradigm. Several studies show that engaging in offensive aggressive behaviors and winning a fight are highly rewarding and/or reinforcing 11. From that perspective, there is no suffering in the resident. However, an aggressive interaction requires an opponent as well. Defensive behavior and submission belongs to the natural repertoire to cope with dominance. Defeat and subjugation triggers an adaptive behavioral and physiological response aimed at adopting a subordinate position in a social group. From that point of view, the initial response during the defeat will lead to a well-adapted animal that does not necessarily suffer 15. Only repeated exposure to a dominant and social isolation after the defeat may lead to a condition that goes beyond the adaptive capacity of the animal. This makes the paradigm suitable as an animal model for stress pathology with a high ecological validity 17. Although the stress of social defeat is mainly of a psychosocial nature, physical harm and injury may occur. In a normal (non-violent) social interaction, this physical harm is limited. Biting occurs mainly at the back and flanks of the opponent; an area of the body with a thick and tough skin 5, 6. Biting is in fact brief nipping of the skin, leaving behind small imprints of the incisors. This type of skin damage does not require any veterinary care. However, biologically functional aggression may change to a pathological, violent form of aggression which is out of control and out of context. In these situations, more serious wounds inflicted in particular at vulnerable body regions (belly, throat and paws) may occur 14. To be clinically relevant, experimental model systems for violent aggressive behavior need to be valid, and this development poses a central ethical dilemma of this type of aggression research, namely harm and injury. Two countervailing principles govern this research: face validity is achieved when the behavior is potentially harmful and injurious, yet, at the same time, every ethical research guideline emphasizes the reduction and avoidance of the risk to be harmed or injured. Each research question and protocol needs to probe how much harm and injury is necessary or acceptable to generate scientifically valid information that can be translated into concerns of the public health system. When research on violence is the main aim of the experiment, it is self-evident that great care should be taken of the victim in terms of wound care or even euthanasia. The presence of serious wounding at vulnerable body regions should be the humanitarian endpoint of the intruder. When social stress is the main aim of the experiment, the interaction should be stopped when the resident shows signs of violence causing serious bite wounds at vulnerable body parts. After all, the psychosocial nature of the stress paradigm should not be mixed with the stress of severe physical injury. When residents show these signs of violence they should be excluded from the experiment.

the resident-intruder paradigm: a standardized test for aggression, violence and social stress

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.

There is a considerable variation between strains and within strains in the level of offensive aggressive behavior. This is demonstrated in Figure 1 which shows the frequency distribution of the offensive aggression score in a laboratory bred but originally feral strain of rats (Wild Type Groningen strain (WTG)) (Figure 1a) and a more common strain of laboratory rats (Wistar, Figure 1b). In the WTG strain, about one third of the animals is extremely aggressive whereas another third is not or very low aggressive. This is in contrast to the frequency distribution of a Wistar strain in which the highly aggressive phenotype is absent and about fifty percent of the animals can be considered as low or non-aggressive 16.
	Figure 2 shows the distribution of different behavioral categories in the resident-intruder paradigm with the WTG strain as residents (Figure 2a) and the Wistar rats as intruders (Figure 2b). Shown is the average composition of offensive behavior in the WTG resident rat and the average composition of defensive behavior in the Wistar intruder in terms of the relative amount of time spent on the various behaviors.
Figure 3 shows an example of the use of the resident-intruder paradigm in behavioral pharmacology. The selective serotonin 1a receptor agonist Alnespirone induces a dose dependent reduction in offensive aggression, which is accompanied by a dose dependent increase in social exploration. The absence of any significant effects on non-social exploration and inactivity supports the view that the behavioral effects of this compound are specific for offensive aggression 9.
In some individuals offensive aggression may escalate into a violent form of aggression. The distinction between high levels of aggression and violence is illustrated in Figure 4. Despite the fact that there is no statistical difference in offense score, the violent form of aggression is characterized by a very short attack latency, attack of an anesthetized male or a female, serious wounding and a very low threat attack ratio 8.
<img src="/files/ftp_upload/4367/4367fig1.jpg" alt="Figure 1" fo:content-width="3in" fo:src="/files/ftp_upload/4367/4367fig1highres.jpg"/> 
Figure 1. Frequency distribution of individual levels of offensive aggression in a laboratory bred, but originally feral rat strain (A - Top panel) and in a Wistar strain of male rats (B - Bottom panel) 16.
<img src="/files/ftp_upload/4367/4367fig2.jpg" alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4367/4367fig2highres.jpg"/> 
Figure 2. Behavioral profile of resident males WTG rats (A) and Wistar intruder rats (B) during a ten minutes resident intruder test.
<img src="/files/ftp_upload/4367/4367fig3.jpg" alt="Figure 3" fo:content-width="4.5in" fo:src="/files/ftp_upload/4367/4367fig3highres.jpg"/> 
Figure 3. Dose dependent reduction of offensive aggression by the 5-HT1a agonist Alnespirone 9. <a href="https://www.jove.com/files/ftp_upload/4367/4367fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/4367/4367fig4.jpg" alt="Figure 4" fo:content-width="2.75in" fo:src="/files/ftp_upload/4367/4367fig4highres.jpg"/> 
Figure 4. Comparison between highly offensive males and violent males 8.

Watch this Scientific Journal Video about The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress at JoVE.com

The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

This video shows the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression, defensive behavior and violence in a semi-natural setting. The use of the paradigm for social stress experiments is explained as well.

This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to ...

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JoVE Journal

Behavior

70.4K 조회수.  University Groningen. 이 동영상은 쥐의 주민 침입자 패러다임을 보여줍니다. 이 테스트는 공격 침략, 반 자연 속에서 방어 행동과 폭력을 측정하는 표준화 된 방법입니다. 사회적 스트레스 실험 패러다임의 사용으로 잘 설명되어 있습니다.

비디오: The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress

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 ...

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 ...

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 ...

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim to a crime may create from memory a holistic facial composite of the culprit with the assistance of a police operator. The aim is that the composite is recognized by someone who believes that they know the culprit. For this paradigm, participants view a culprit actor on video and following a delay, participant-witnesses construct a holistic system facial composite. Controls do not construct a composite. From a series of arrays of computer-generated, but realistic faces, the holistic system construction method primarily requires participant-witnesses to select the facial images most closely meeting their memory of the culprit. Variation between faces in successive arrays is reduced until ideally the final image possesses a close likeness to the culprit. Participant-witness directed tools can also alter facial features, configurations between features and holistic properties (e.g., age, distinctiveness, skin tone), all within a whole face context. The procedure is designed to closely match the holistic manner by which humans&#8217; process faces. On completion, based on their memory of the culprit, ratings of composite-culprit similarity are collected from the participant-witnesses. Similar ratings are collected from culprit-acquaintance assessors, as a marker of composite recognition likelihood. Following a further delay, all participants &#8212; including the controls&#160;&#8212; attempt to identify the culprit in either a culprit-present or culprit-absent video line-up, to replicate circumstances in which the police have located the correct culprit, or an innocent suspect. Data of control and participant-witness line-up outcomes are presented, demonstrating the positive influence of holistic composite construction on identification accuracy. Correlational analyses are conducted to measure the relationship between assessor and participant-witness composite-culprit similarity ratings, delay, identification accuracy, and confidence to examine which factors influence video line-up outcomes.

This applied experimental paradigm replicates circumstances by which an eyewitness to a real crime may create a holistic facial composite of the culprit from memory, and then attempt to identify the culprit from a video line-up containing the culprit or one in which he or she is not present.

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

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 HoneyComb Paradigm for Research on Collective Human Behavior

Collective human behavior such as group movement frequently shows surprising patterns and regularities, such as the emergence of leadership. Recent literature has revealed that these patterns, often visible at the global level of the group, are based on self-organized, individual behaviors that follow several simple local parameters. Understanding the dynamics of human collective behavior can help to improve coordination and leadership in group and crowd scenarios, such as identifying the ideal placement and number of emergency exits in buildings.
In this article, we present the experimental paradigm HoneyComb, which can be used to systematically investigate conditions and effects of human collective behavior. This paradigm uses a computer-based multi-user platform, providing a setting that can be shaped and adapted to various types of research questions. Situational conditions (e.g., cost-benefit ratios for specific behavior, monetary incentives and resources, various degrees of uncertainty) can be set by experimenters, depending on the research question. Each participant's motions are recorded by the server as hexagonal coordinates with timestamps at an accuracy of 50 ms and with individual IDs. Thus, a metric can be defined on the playfield, and movement parameters (e.g., distances, velocity, clustering, etc.) of participants can be measured over time. Movement data can in turn be combined with non-computerized data from questionnaires garnered within the same experiment setup.
The HoneyComb paradigm is paving the way for new types of human movement experiments. We demonstrate here that these experiments can render results with sufficient internal validity to meaningfully deepen our understanding of human collective behavior.

Here, we present the computer-based, multi-agent game HoneyComb, which enables experimental investigations of collective human movement behavior via black-dot-avatars on a virtual 2D hexagonal playfield. Different experimental conditions, like variable&#160;incentives on goal fields or vision radius, can be set, and their effects on human movement behavior can be investigated.

Collective human behavior such as group movement frequently shows surprising patterns and regularities, such as the emergence of leadership. Recent ...

Enactive Phenomenological Approach to the Trier Social Stress Test: A Mixed Methods Point of View

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. These tests are based on uncontrollability and social-evaluative threat, inducing psychological and physiological consequences (e.g., anxiety, emotional states, salivary cortisol increases). Many quantitative experimental studies have investigated these stress inducers and these consequences. But, as far as we know, this study is the first to provide a qualitative analysis to access the participants' voices so as to understand the dynamics of their experience throughout the TSST and the TSST-G. This paper outlines a mixed methods approach to the TSST. This approach can help to maximize the information that can be gained from the TSST, which researchers often use without looking more closely at what is qualitatively happening psychologically for participants during the stressor itself. In this way, this protocol is an example of mixed methods, showing the added value of using the enactive phenomenological approach to analyze experimental protocols more deeply. This kind of mixed methods is helpful to access the experience, to understand the actor's point of view, and to analyze in-depth the dynamics of cognitive processes like intentions, perceptions, enacted knowledge, and emotion. The discussion section shows the different uses of a mixed methods protocol, exploiting the enactive phenomenological approach to analyze a protocol or to give a cross vision of the same research subject. This section deals with different existing applications, pointing out some critical steps in this mixed methods approach.

Here, we present an original mixed method combining a quantitative and a qualitative approach to examine accurately participants&#39; experiences during the Trier Social Stress Test and the Trier Social Stress Test for groups.

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. ...

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 ...

Assessment of Stress Effects on Cognitive Flexibility using an Operant Strategy Shifting Paradigm

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and duration of the stressor; 2) type of cognitive function under study; and 3) timing of the stressor in relation to learning or executing the cognitive task. Furthermore, sex differences among the effects of stress on cognitive function have been widely documented. Described here is an adaptation of an automated operant strategy shifting paradigm to assess how variations in stress affect cognitive flexibility in male and female Sprague Dawley rats. Specifically, restraint stress is used before or after training in this operant-based task to examine how stress affects cognitive performance in both sexes. Particular brain areas associated with each task in this automated paradigm have been well-established (i.e., the medial prefrontal cortex and orbitofrontal cortex). This allows for targeted manipulations during the experiment or the assessment of particular genes and proteins in these regions upon completion of the paradigm. This paradigm also allows for the detection of different types of performance errors that occur after stress, each of which has defined neural substrates. Also identified are distinct sex differences in perseverative errors after a repeated restraint stress paradigm. The use of these techniques in a preclinical model may reveal how stress affects the brain and impairs cognition in psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), which display marked sex differences in prevalence.

Stressful life events impair cognitive function, increasing the risk of psychiatric disorders. This protocol illustrates how stress affects cognitive flexibility using an automated operant strategy shifting paradigm in male and female Sprague Dawley rats. Specific brain areas underlying particular behaviors are discussed, and translational relevance of results are explored.

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and ...

A Standardized Method for Measurement of Elbow Kinesthesia

Proprioception is an important component of controlled movement. The threshold to detection of passive movement (TDPM) is a commonly used method for quantifying the proprioceptive submodality of kinesthesia in research settings. The TDPM paradigm has been found to be valid and reliable; however, the equipment and methods used for TDPM vary between studies. In particular, the research laboratory apparatuses for producing passive movement of an extremity are often custom designed by individual laboratories or inaccessible due to high cost. There is a need for a standardized, valid, and reliable method for measuring TDPM using readily available equipment. The purpose of this protocol is to provide a standardized method for measurement of TDPM at the elbow that is economical, easy to administer, and that produces quantitative results for measurement purposes in research-based settings. This method was tested on 20 healthy adults without neurological impairment, and eight adults with chronic stroke. The results obtained suggest this method is a reliable way to quantify elbow TDPM in healthy adults, and provides initial support for validity. Researchers seeking a balance between equipment affordability and measurement precision are most likely to find this protocol of benefit.

Here, we present a standardized method for measurement of elbow passive kinesthesia using the threshold to detection of passive movement (TDPM) that is appropriate for a research setting.

Proprioception is an important component of controlled movement. The threshold to detection of passive movement (TDPM) is a commonly used method for ...