This research is supported by the Collaborative Research Center 1193, Subproject Z02, funded by the German National Research Foundation (SFB1193, Neurobiology of Resilience) and the Boehringer Ingelheim Foundation (grant to Leibniz Institute for Resilience Research and Individual Phenotyping and High-Resolution Automated Behavioural Analysis). We would like to thank Dr. Konstantin Radyushkin and Mrs. Sandra Reichel for their technical assistance as well as Mrs. Hanna Kim for her English language support. The funding sources had no involvement in the model design; collection, analysis, and interpretation of data; in the writing of the protocol; and in the decision to submit the protocol for publication.

All procedures were performed in accordance with the European Communities Council Directive regarding the care and use of animals for experimental procedures and were approved by local authorities (Landesuntersuchungsamt Rheinland-Pfalz). Figure 1 represents a schematic timeline.
1. Treatment
<ol>
	<li>Animals of interest: Obtain C57BL6/J male mice at 7 weeks of age, and upon arrival, single-house in a temperature- and humidity-controlled facility on a 12 h light-dark cycle (lights on: 8:00; lights off: 20:00; 23 &#176;C; 38% humidity) with food and water ad libitum.</li>
	<li>Chronic social defeat (CSD)
	<ol>
		<li>Treatment group
		<ol>
			<li>Following 1 week of habituation, perform CSD treatment for 10 consecutive days using the CD-1 strain as the resident&#39;s strain (for a detailed protocol, refer to chronic social defeat9 and the modified chronic social defeat treatment10).</li>
			<li>Introduce the C57BL6/J mouse into the cage of the CD-1 mouse and count 10 s of physical attack. Repeat this episode three times, each with a different CD-1 mouse, and separate by 15 min inter-episode intervals.</li>
			<li>Place a mesh wall between the C57BL6/J mouse and CD-1 mouse during these intervals, allowing only sensory contact. Following the third episode, house the C57BL6/J mice overnight in the cages of the CD-1 mice, separating both by a mesh wall. Repeat for 10 days. 
			NOTE: CD-1 mice number = C57BL6/J mice number + 1. If the number of C57BL6/J treated mice is below 10 then a minimum of 10 CD-1 mice is still needed to ensure that every day the last sensory phase (lasting overnight) is with a new CD-1 mouse throughout the 10 days of treatment.</li>
			<li>Carefully assess the physical well-being of the animals throughout the 10 days. If an animal is severely wounded, exclude it from the experiment for ethical and scientific (mobility/activity during the post-treatment test) reasons. Table 1 provides a well-being checklist.</li>
		</ol>
		</li>
		<li>Control group
		<ol>
			<li>Upon arrival, maintain same-age mice in the same conditions as the treatment group.</li>
			<li>Following 1 week of habituation, introduce the control animals for 90 s in an empty cage and then return them to individual cages (single-housed) separated in half by mesh walls identical to those used for the treatment group. Perform this daily in parallel to the 10 treatment days. 
			NOTE: It is advisable to keep the control group and the treatment group housed in different rooms.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After the last (10th) sensory phase, single-house all mice in new cages in similar conditions to those described upon arrival and leave them to rest overnight. 
	NOTE: The last sensory phase should last 24 h, then animals are single-housed.</li>
</ol>
2. Post-treatment test: Social threat-safety test (Figure 2)
<ol>
	<li>Following CSD treatment, single-house all mice (treated and control groups) in new cages in similar conditions to those described upon arrival and leave them to rest overnight.</li>
	<li>During the morning hours (8:00-13:30), clean the three-chambered arena (rectangle in shape with a total size of 60 cm x 40 cm, made of transparent acrylic walls and smooth floors) with 5% ethanol and place it under the camera with light conditions of 37 lux. Ensure that the entire arena is visible.</li>
	<li>Clean the mesh enclosures (cage-like made from metal or acrylic) with 5% ethanol and position them as shown in the corners in Figure 1A.</li>
	<li>Habituation phase: Introduce the animal of interest at the center of the arena, allow for exploration for 6 min, and then return them to their home cage.</li>
	<li>Place the novel (unknown) CD-1 social target (conspecific) under one mesh enclosure and the novel 129/Sv social target under the other mesh enclosure. 
	NOTE: It is important to use an unknown 129/Sv conspecific to avoid a familiarity bias. Preferably have 4 mesh enclosures per arena: 2 attributed to the habituation phase and 2 to the testing phase.</li>
	<li>Testing phase: Immediately re-introduce the animal of interest at the center of the arena and allow exploration for 6 min.</li>
	<li>Return all animals to their home. Clean the arena and mesh enclosures with 5% ethanol between tests of different animals, but never during the observation of the same animal, i.e., never between habituation and testing phases.</li>
	<li>Alternate the location of the mesh enclosures between animals (and never between the two phases within the same animal) to control for possible location preferential bias.</li>
</ol>
3. Scoring and analysis
NOTE: Only the post-stress treatment test, i.e., the STST is scored and analyzed (and not the CSD stress treatment).
<ol>
	<li>Define the interaction zone as 2 cm around the mesh enclosures&#39; boundaries.</li>
	<li>Score the duration spent exploring the mesh enclosures during the habituation phase when the animal&#39;s nose is within the interaction zone.</li>
	<li>Score the duration spent interacting with the social targets during the testing phase when the animal&#39;s nose is within the interaction zone. 
	NOTE: Detection can be achieved either manually (using a timer or software for manual scoring) or automatically. Regardless of the detection method, take the nose point for exploration and social interaction measurements and the center point of the body for activity-related measurements (e.g., distance moved).</li>
	<li>Calculate the social interaction index as follows: time spent exploring each social target during the testing phase / average time spent exploring the two empty mesh enclosures during the habituation phase (Figure 2B).</li>
	<li>Divide the treatment group into 3 subgroups as follows: Animals with a social interaction index &#8805;1 with the CD-1 social target are non-avoiders, animals with a social interaction index &#60;1 with both social targets are indiscriminate-avoiders, animals with a social interaction index &#8805;1 only with the 129/Sv social target are discriminating-avoiders (Figure 2C-D). 
	NOTE: The number of animals within each of the three subgroups can differ between different animal batches (about 1/3 of all animals that undergo the CSD treatment will display the phenotypic characteristics of one of the three subgroups).</li>
	<li>Assess the stress effect by statistically analyzing the social interaction index with the CD-1 social target between the treatment and control groups (either parametric two-sample t-test or nonparametric Mann-Whitney test).</li>
</ol>

Threat-Safety Test for Stress Susceptibility or Resilience in Mice Models

<ol>
	<li>Hyman, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mice+cope+with+stressful+social+situations.">How mice cope with stressful social situations.</a> Cell. 131 (2), 232-234 (2007).</li><li>Kessler, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+stressful+life+events+on+depression.">The effects of stressful life events on depression.</a> Annual Review of Psychology. 48 (1), 191-214 (1997).</li><li>Vos, T., 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=Global,+regional,+and+national+incidence,+prevalence,+and+years+lived+with+disability+for+301+acute+and+chronic+diseases+and+injuries+in+188+countries,+1990-2013:+A+systematic+analysis+for+the+Global+Burden+of+Disease+Study,+2013.">Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: A systematic analysis for the Global Burden of Disease Study, 2013.</a> The Lancet. 386 (9995), 743-800 (2015).</li><li>Bj&#246;rkqvist, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+defeat+as+a+stressor+in+humans.">Social defeat as a stressor in humans.</a> Physiology &amp; Behavior. 73 (3), 435-442 (2001).</li><li>Blanchard, R. J., McKittrick, C. R., 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=Animal+models+of+social+stress:+effects+on+behavior+and+brain+neurochemical+systems.">Animal models of social stress: effects on behavior and brain neurochemical systems.</a> Physiology &amp; Behavior. 73 (3), 261-271 (2001).</li><li>Singleton, G. R., Krebs, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3-+The+Secret+World+of+Wild+Mice.+The+Mouse+in+Biomedical+Research.">Chapter 3- The Secret World of Wild Mice. The Mouse in Biomedical Research.</a> American College of Laboratory Animal Medicine. The Mouse in Biomedical Research (Second Edition). 1, 25-51 (2007).</li><li>Kondrakiewicz, K., Kostecki, M., Szadzi&#324;ska, W., Knapska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+validity+of+social+interaction+tests+in+rats+and+mice.">Ecological validity of social interaction tests in rats and mice.</a> Genes, Brain, and Behavior. 18 (1), e12525 (2019).</li><li>Martinez, M., Calvo-Torrent, A., Pico-Alfonso, M. 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+defeat+and+subordination+as+models+of+social+stress+in+laboratory+rodents:+a+review.">Social defeat and subordination as models of social stress in laboratory rodents: a review.</a> Aggressive Behavior: Official Journal of the International Society for Research on Aggression. 24 (4), 241-256 (1998).</li><li>Golden, S. A., Covington III, 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> Nature Protocols. 6 (8), 1183 (2011).</li><li>van der Kooij, M. 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=Chronic+social+stress-induced+hyperglycemia+in+mice+couples+individual+stress+susceptibility+to+impaired+spatial+memory.">Chronic social stress-induced hyperglycemia in mice couples individual stress susceptibility to impaired spatial memory.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (43), E10187-E10196 (2018).</li><li>Chartier, M. J., Walker, J. R., Stein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considering+comorbidity+in+social+phobia.">Considering comorbidity in social phobia.</a> Social Psychiatry and Psychiatric Epidemiology. 38 (12), 728-734 (2003).</li><li>Cohen, H., Zohar, J., Matar, M. A., Kaplan, Z., Geva, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsupervised+fuzzy+clustering+analysis+supports+behavioral+cutoff+criteria+in+an+animal+model+of+post-traumatic+stress+disorder.">Unsupervised fuzzy clustering analysis supports behavioral cutoff criteria in an animal model of post-traumatic stress disorder.</a> Biological Psychiatry. 58 (8), 640-650 (2005).</li><li>Scharf, S. H., Schmidt, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+stress+vulnerability+and+resilience+in+translational+research.">Animal models of stress vulnerability and resilience in translational research.</a> Current Psychiatry Reports. 14 (2), 159-165 (2012).</li><li>Krishnan, V., 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=Molecular+adaptations+underlying+susceptibility+and+resistance+to+social+defeat+in+brain+reward+regions.">Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions.</a> Cell. 131 (2), 391-404 (2007).</li><li>Ayash, S., Schmitt, U., M&#252;ller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+social+defeat-induced+social+avoidance+as+a+proxy+of+stress+resilience+in+mice+involves+conditioned+learning.">Chronic social defeat-induced social avoidance as a proxy of stress resilience in mice involves conditioned learning.</a> Journal of Psychiatric Research. 120, 64-71 (2020).</li><li>Ayash, 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=Fear+circuit-based+neurobehavioural+signatures+mirror+resilience+to+chronic+social+stress+in+mouse.">Fear circuit-based neurobehavioural signatures mirror resilience to chronic social stress in mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 120 (17), e2205576120 (2023).</li><li>Duits, P., 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=Updated+meta-analysis+of+classical+fear+conditioning+in+the+anxiety+disorders.">Updated meta-analysis of classical fear conditioning in the anxiety disorders.</a> Depression and Anxiety. 32 (4), 239-253 (2015).</li><li>Coifman, K. G., Bonanno, G. A., Rafaeli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affect+dynamics,+bereavement,+and+resilience+to+loss.">Affect dynamics, bereavement, and resilience to loss.</a> Journal of Happiness Studies. 8 (3), 371-392 (2007).</li><li>Waugh, C. E., Thompson, R. J., Gotlib, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+emotional+responsiveness+in+trait+resilience.">Flexible emotional responsiveness in trait resilience.</a> Emotion. 11 (5), 1059 (2011).</li><li>Bonanno, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss,+trauma,+and+human+resilience:+Have+we+underestimated+the+human+capacity+to+thrive+after+extremely+aversive+events.">Loss, trauma, and human resilience: Have we underestimated the human capacity to thrive after extremely aversive events.</a> American Psychologist. 59 (1), 20 (2004).</li><li>Bonanno, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resilience+in+the+face+of+potential+trauma.">Resilience in the face of potential trauma.</a> Current Directions in Psychological Science. 14 (3), 135-138 (2005).</li><li>Yehuda, R., Flory, J. D., Southwick, S., Charney, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+an+agenda+for+translational+studies+of+resilience+and+vulnerability+following+trauma+exposure.">Developing an agenda for translational studies of resilience and vulnerability following trauma exposure.</a> Annals of the New York Academy of Sciences. 1071 (1), 379-396 (2006).</li><li>Grillon, C., Morgan III, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear-potentiated+startle+conditioning+to+explicit+and+contextual+cues+in+Gulf+War+veterans+with+post-traumatic+stress+disorder.">Fear-potentiated startle conditioning to explicit and contextual cues in Gulf War veterans with post-traumatic stress disorder.</a> Journal of Abnormal Psychology. 108 (1), 134 (1999).</li><li>Brewin, C. 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+cognitive+neuroscience+account+of+post-traumatic+stress+disorder+and+its+treatment.">A cognitive neuroscience account of post-traumatic stress disorder and its treatment.</a> Behaviour Research and Therapy. 39 (4), 373-393 (2001).</li><li>Milad, M. R., Rauch, S. L., Pitman, R. K., Quirk, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+extinction+in+rats:+implications+for+human+brain+imaging+and+anxiety+disorders.">Fear extinction in rats: implications for human brain imaging and anxiety disorders.</a> Biological Psychology. 73 (1), 61-71 (2006).</li><li>Jovanovic, T., Norrholm, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+impaired+fear+inhibition+in+post-traumatic+stress+disorder.">Neural mechanisms of impaired fear inhibition in post-traumatic stress disorder.</a> Frontiers in Behavioral Neuroscience. 5, 44 (2011).</li><li>Morton, D. B., Griffiths, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+the+recognition+of+pain,+distress+and+discomfort+in+experimental+animals+and+an+hypothesis+for+assessment.">Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment.</a> The Veterinary Record. 116 (6), 431-436 (1985).</li><li>Goldsmith, J. F., Brain, P. F., Benton, D. <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+the+duration+of+individual+or+group+housing+on+behavioural+and+adrenocortical+reactivity+in+male+mice.">Effects of the duration of individual or group housing on behavioural and adrenocortical reactivity in male mice.</a> Physiology &amp; Behavior. 21 (5), 757-760 (1978).</li><li>Cairns, R. B., Hood, K. E., Midlam, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+fighting+in+mice:+Is+there+a+sensitive+period+for+isolation+effects.">On fighting in mice: Is there a sensitive period for isolation effects.</a> Animal Behaviour. 33 (1), 166-180 (1985).</li><li>Varlinskaya, E. I., Spear, L. P., Spear, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+behavior+and+social+motivation+in+adolescent+rats:+role+of+housing+conditions+and+partner's+activity.">Social behavior and social motivation in adolescent rats: role of housing conditions and partner's activity.</a> Physiology &amp; Behavior. 67 (4), 475-482 (1999).</li><li>Sial, O. K., Warren, B. L., Alcantara, L. F., Parise, E. M., Bola&#241;os-Guzm&#225;n, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vicarious+social+defeat+stress:+Bridging+the+gap+between+physical+and+emotional+stress.">Vicarious social defeat stress: Bridging the gap between physical and emotional stress.</a> Journal of Neuroscience Methods. 258, 94-103 (2016).</li><li>Brown, R. E., Brown, R. E., Macdonald, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rodents II. Suborder Myomorpha. [In: Social Odours in Mammals]. 1, (1985).</li><li>Haney, M., 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=Ultrasounds+during+agonistic+interactions+between+female+rats+(Rattus+norvegicus).">Ultrasounds during agonistic interactions between female rats (Rattus norvegicus).</a> Journal of Comparative Psychology. 107 (4), 373 (1993).</li><li>Warren, B. L., 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=Neurobiological+sequelae+of+witnessing+stressful+events+in+adult+mice.">Neurobiological sequelae of witnessing stressful events in adult mice.</a> Biological Psychiatry. 73 (1), 7-14 (2013).</li><li>Malatynska, E., Knapp, 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=Dominant-submissive+behavior+as+models+of+mania+and+depression.">Dominant-submissive behavior as models of mania and depression.</a> Neuroscience &amp; Biobehavioral Reviews. 29 (4-5), 715-737 (2005).</li><li>Avgustinovich, D. F., Kovalenko, I. L., Kudryavtseva, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+anxious+depression:+persistence+of+behavioral+pathology.">A model of anxious depression: persistence of behavioral pathology.</a> Neuroscience and Behavioral Physiology. 35 (9), 917-924 (2005).</li><li>Vennin, 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=A+resilience+related+glial-neurovascular+network+is+transcriptionally+activated+after+chronic+social+defeat+in+male+mice.">A resilience related glial-neurovascular network is transcriptionally activated after chronic social defeat in male mice.</a> Cells. 11 (21), 3405 (2022).</li><li>Ayash, S., Schmitt, U., Lyons, D. M., M&#252;ller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+inoculation+in+mice+induces+global+resilience.">Stress inoculation in mice induces global resilience.</a> Translational Psychiatry. 10 (1), 200 (2020).</li><li>Yuan, R., 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=Long-term+effects+of+intermittent+early+life+stress+on+primate+prefrontal-subcortical+functional+connectivity.">Long-term effects of intermittent early life stress on primate prefrontal-subcortical functional connectivity.</a> Neuropsychopharmacology. 46 (7), 1348-1356 (2021).</li><li>Lyons, D. M., Ayash, S., Schatzberg, A. F., M&#252;ller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+validity+of+social+defeat+stressors+in+mouse+models+of+vulnerability+and+resilience.">Ecological validity of social defeat stressors in mouse models of vulnerability and resilience.</a> Neuroscience &amp; Biobehavioral Reviews. 145, 105032 (2023).</li><li>Oizumi, 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=Influence+of+aging+on+the+behavioral+phenotypes+of+C57BL/6J+mice+after+social+defeat.">Influence of aging on the behavioral phenotypes of C57BL/6J mice after social defeat.</a> PLoS One. 14 (9), e0222076 (2019).</li></ol>

The authors have nothing to disclose.

The behavioral protocol here describes the Social Threat-Safety Test, used to divide a single group post-CSD treatment into three different subgroups, serving as a method to investigate the underlying biology of stress susceptibility and resilience and to test potential therapies. The biological context and technical details need to be carefully considered to guide a thorough experimental design.
Different housing conditions can alter aggression sociability levels, potentially influencing resu...

Stress is defined as the disruption of homeostasis caused by physical or psychological stimuli1. Stress is a well-known major risk factor for the development of mental disorders such as post-traumatic stress disorder, depression, and anxiety2,3. In particular, social stress is considered a major risk factor for the development of stress-related mental disorders4. One type of social stress that has gained particular importance in research is social subordination stress5. Mice, like humans, are capable of a rich set of social behavior6, rendering them suitable for investigations involving social stress. In the laboratory setting, when adult mice are group housed, they establish a social structure involving the formation of ranks7. Accordingly, the colony model was designed to study the effects of naturally established social hierarchies in mixed-sex groups of mice8. Over the years, variations of the colony model have been developed to utilize social subordination stress, including same-sex group models, the social instability model, and the intruder-colony model. In recent years, however, one particular variant known as the male resident-intruder model has been popularized in literature, simplifying the social complexity to two mice: a resident and an intruder. The animal of interest, known as the intruder, is placed into the cage of a larger, older, and retired breeder, known as the resident or aggressor. The resident then physically attacks the intruder as a method of confrontation, establishing a social hierarchy wherein the resident is dominant and the intruder is subordinate. When confrontations are one-time events, they classify as &#34;acute&#34; (the &#34;acute social defeat model&#34;), whereas repeated confrontations lasting over several days (usually 10) are known as &#34;chronic&#34; (the &#34;chronic social defeat model&#34;). In the chronic social defeat (CSD) model, attacks are intermittent and typically confined to a period of 5-10 min9, termed the physical phase. Following the physical phase, the intruder and resident are kept overnight in the same cage, separated in half with a mesh wall, allowing for all forms of interaction except physical contact. This configuration, known as the sensory phase, induces stress through the continuous appearance of threat instead of direct physical confrontation. In 2018, van der Kooji and colleagues introduced a modified chronic social defeat treatment to focus on the psychosocial component of the model by standardizing and strictly limiting the physical phase10. The modified model limits physical attacks to three 10 s episodes with different residents, occurring in 15 min inter-episode intervals of the sensory phase. Following the third physical episode, the sensory phase lasts overnight. This cycle repeats for 10 consecutive days with new residents per episode. The modified treatment enhances the translational validity of the chronic social defeat model as physical harm of the intruder is minimized, and outcome variability from differential durations of physical attacks is reduced.
Since the CSD model is utilized to study stress-related illness (e.g., depression, anxiety, post-traumatic stress disorder), post-behavioral assays are chosen, including, but not limited to, behavioral assays of aggression, memory, and anhedonia. In recent years, post-CSD behavioral assays in mice often evaluate how and to what extent sociability is affected9. Sociability is defined as the innate preference of mice to socially interact rather than socially avoid a conspecific. Since sociability is subject to stress effects, assays that solely assess social avoidance development were established. Stress-induced social avoidance has a translational relevance as it represents one of the main behavioral symptoms of social anxiety and depression in humans11. Similar to humans, not all mice develop social avoidance following CSD treatment, suggesting the presence of individuality in stress responsiveness. Cohen and colleagues have proposed cut-off behavioral criteria to be a promising approach for studying the neurobiology of individuality12. Selection of animals based on behavior results in group division, underlining the basis for gene-environment studies. Subsequently, different subgroups often show distinct enrichment of specific genetic variants/modifications, which in turn can be investigated under different environmental conditions13. Accordingly, individuality in the development of social avoidance was utilized to divide the single group of chronically socially defeated male mice into two subgroups: stress susceptible (socially avoidant) and stress resilient (socially non-avoidant9,14). However, the interpretation of the social avoidance phenotype in mice as a maladaptive or adaptive behavior should be considered in the overall context of both the treatment (here CSD) and post-treatment behavioral assay. Additionally, the post-treatment behavioral assay of choice would ideally assess other facets of sociability and not solely social avoidance development. Our recent work revealed the involvement of conditioned learning in CSD-induced social avoidance15. Specifically, CSD-induced social avoidance is an aversive conditioned response towards the characterizing traits of the residents&#39; strain serving as the conditioned stimulus to the unconditioned stimulus, namely the attacks by the residents. Moreover, within the socially avoidant subgroup, some individuals can discriminate between the traits of the aversive residents&#39; strain and those of other safe novel strains, while other individuals show generalized social avoidance to both strains. We propose here a refined behavioral post-CSD assay: the Social Threat-Safety Test (STST)15. Unlike other social interaction tests9, the STST enables a simultaneous assessment of social avoidance development as a measurement of the correct aversive conditioned response (i.e., successful conditioned learning) and social threat-safety discrimination ability, both of which are utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice. The assessment of social threat-safety discrimination versus aversive response generalization extends the translational criteria used to classify the single group of chronically socially defeated animals into resilient and susceptible subgroups.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arenas</td>
			<td>Noldus, Sociability cage, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/applications/sociability-cage</td>
			<td>Three-chambered, rectangle in shape with a total size of 60 cm x&#160; 40 cm, made of acrylic transparent walls and smooth floors</td>
		</tr>
		<tr>
			<td>Camera for video recording</td>
			<td>Basler AG, Germany 
			An der Strusbek 60-62 
			22926 Ahrensburg</td>
			<td>&#160;ace Classic 
			acA1300-60gc</td>
			<td>If using automatic detection program, make sure cameras are compatible</td>
		</tr>
		<tr>
			<td>Camera objective</td>
			<td>KOWA Kowa Optimed Deutschland GmbH 
			Fichtenstr. 123 
			40233 Duesseldorf: LMVZ4411 | 1/1.8&#34; 4.4~11mm Varifokal Objektiv</td>
			<td>Part-No. 10504</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection program/Timer&#160;</td>
			<td>Noldus, EthoVision-XT, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/ethovision-xt</td>
			<td>Detection can be achieved either manually (using a timer or a software for manual scoring) or automatically</td>
		</tr>
		<tr>
			<td>Housing cages</td>
			<td>ZOONLAB GmbH, Hermannstra&#223;e 6, 
			44579 Castrop-Rauxel</td>
			<td>3010010</td>
			<td>Type 2 cages: 265 mm x 205 mm x 140 mm (l x w x h) i.e. 360 cm&#178; bottom area. Made of Polycarbonate (Makrolone&#169;) and Polysulfone. Lids are made of stainless steel. European standard cages for up to 5 mice (20&#8211;25 g). Autoclavable up to 134 &#176;C</td>
		</tr>
		<tr>
			<td>Mesh enclosures&#160;</td>
			<td>Part of the Arena Package: Noldus, Sociability cage, Wageningen, the Netherlands</td>
			<td>https://www.noldus.com/applications/sociability-cage</td>
			<td>Small acrylic or metal cage-like with a diameter of 100 mm and a height of 200 mm with openings of a 10 mm in size. Two mesh enclosures per arena would work but four is preferable (see point 2.5 in protocol)</td>
		</tr>
		<tr>
			<td>Mesh wall</td>
			<td>selfmade</td>
			<td>N/A</td>
			<td>Acrylic or metal, one for each cage. Size depends on cages used. The walls must not allow the two animals to have a physical contact</td>
		</tr>
		<tr>
			<td>Social targets: Mice of the strains CD-1 and 129/Sv; retired male breeders</td>
			<td>Mice provided by Charles River: 
			Strain name: CD-1&#174;IGS Mouse 
			129S2/SvPasCrl&#160;</td>
			<td>Crl:CD1(ICR); 129S2/SvPasCrl&#160;</td>
			<td>CD-1 and 129/Sv retired male breeders, single-housed, novel (unknown) conspecifics to the animals of interest. If retired male breeders are not available then males older than 1 year from both strains would suffice</td>
		</tr>
	</tbody>
</table>

social threat-safety test uncovers psychosocial stress-related phenotypes

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience and its behavioral impact on mice should be comparable to humans. Chronic social defeat (CSD) utilizes a type of social stress involving physical attacks and sensory threats to induce mental dysfunctions resembling human affective disorders. To strengthen the psychosocial component of CSD, a 10-day CSD protocol was applied in which daily physical attacks are standardized to three 10 s episodes followed by a 24 h sensory phase. After the 10th sensory phase, the CSD protocol is followed by a refined behavioral assay called the social threat-safety test (STST). Post-stress behavioral assays need to determine how and to what extent the social stressor has influenced behavior. The STST allows chronically socially defeated male mice to interact with 2 novel male individuals (social targets): one social target from the attacking strain encountered during the CSD days and the other from a novel strain. Both are presented simultaneously in different compartments of a three-chambered test arena. The test enables a simultaneous assessment of social avoidance development to measure successful aversive conditioned learning and social threat-safety discrimination ability. The development of social avoidance towards both strains reflects a generalized aversive response and thus, a measurement of stress susceptibility. Meanwhile, the development of social avoidance towards only the attacking strain reflects threat-safety discrimination and thus, a measurement of stress resilience. Finally, the absence of social avoidance towards the attacking strain reflects impaired aversive conditioned learning. The protocol aims to refine the currently used mouse models of stress susceptibility/resilience by including translational criteria, specifically threat-safety discrimination and aversive response generalization, to categorize a single group of chronically socially defeated animals into resilient and susceptible subgroups, eventually advancing future translational approaches.

Social interaction index as a measurement of aversive conditioned response 
A social interaction index &#8805;1 reflects greater social interaction with the respective social target compared to the exploration of the empty mesh enclosures. Under baseline conditions, defined here as having neither appetitive nor aversive experience with the characterizing traits of a specific strain (here both social targets to the control group and the 129/Sv social target to the treatment group), intact sociability...

Watch this Scientific Journal Video about Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research at JoVE.com

Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Social Threat-Safety Test Uncovers Psychosocial Stress-Related Phenotypes

Social stress is a major cause of the development of mental disorders. To enhance the translational value of preclinical studies, social stress experience ...

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Research

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Behavior

3.6K Views.  Leibniz-Institute for Resilience Research. The social threat-safety test allows a simultaneous assessment of social avoidance development as a measurement of aversive conditioned learning and social threat-safety discrimination ability, both utilized to identify stress-susceptible and stress-resilient individuals within a single group of chronically socially defeated male mice.

Video: Author Spotlight: Unveiling Mechanisms of Stress Resilience - Significant Findings, Advancements, and Future Research

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.

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 ...

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device (ALDM) Test Systems

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys maintained in semi-free ranging conditions. Data accumulated during the last five years have consistently demonstrated the efficiency of this protocol to investigate individual/physical cognition in monkeys, and have further shown that this procedure reduces stress level during animal testing3. This paper demonstrates that networks of ALDM can also be used to investigate different facets of social cognition and in-group expressed behaviors in monkeys, and describes three illustrative protocols developed for that purpose. The first study demonstrates how ethological assessments of social behavior and computerized assessments of cognitive performance could be integrated to investigate the effects of socially exhibited moods on the cognitive performance of individuals. The second study shows that batteries of ALDM running in parallel can provide unique information on the influence of the presence of others on task performance. Finally, the last study shows that networks of ALDM test units can also be used to study issues related to social transmission and cultural evolution. Combined together, these three studies demonstrate clearly that ALDM testing is a highly promising experimental tool for bridging the gap in the animal literature between research on individual cognition and research on social cognition.

Assessment of Social Cognition in Non-human Primates Using a Network of Computerized Automated Learning Device ALDM Test Systems

Fagot &#38; Paleressompoulle1 have published an automated learning device (ALDM) aimed at testing individual cognitive abilities in semi-free ranging monkeys. The main goal of our protocol is to use a network of ALDM test units to study social cognition in non-human primates.

Fagot &#38; Paleressompoulle1 and Fagot &#38; Bonte2 have published an automated learning device (ALDM) for the study of cognitive abilities of monkeys ...

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 ...

Experimental Research Examining How People Can Cope with Uncertainty Through Soft Haptic Sensations

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has focused on the cognitive strategies people adopt to deal with uncertainty. However, especially when uncertainty is due to unpredictable societal events (e.g., economical crises, political revolutions, terrorism threats) of which one is unable to judge the impact on one&#8217;s future live, cognitive strategies (like seeking additional information) is likely to fail to combat uncertainty. Instead, the current paper discusses a method demonstrating that people might deal with uncertainty experientially through soft haptic sensations. More specifically, because touching something soft creates a feeling of comfort and security, people prefer objects with softer as compared to harder properties when feeling uncertain. Seeking for softness is a highly efficient and effective tool to deal with uncertainty as our hands are available at all times. This protocol describes a set of methods demonstrating 1) how environmental (un)certainty can be situationally activated with an experiential priming procedure, 2) that the quality of the softness experience (what type of softness and how it is experienced) matters and 3) how uncertainty can be reduced using different methods.

To date research has focused on cognitive strategies people adopt to cope with uncertainty. This research examines instead an experiential way of dealing with uncertainty and introduces a set of experimental methods showing how the experience of haptic softness can serve as a tool to deal with uncertainty.

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has ...

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 ...

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...

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. ...