The authors thank Drs. Kentaro Nagaoka (Tokyo University of Agriculture and Technology) and Wataru Iio (Ibaraki Prefecture) for the helpful discussion. This research was supported in part by Ibaraki University Cooperation between Agriculture and Medical Science (IUCAM) (The MEXT, Japan) and the Research Project on the Development of Agricultural Products and Foods with Health-promoting Benefits (NARO) (The MAFF, Japan).

The animal studies were approved by and met the guidelines of both the Animal Care and Use Committee of Ibaraki University and the Ministry of Education Culture, Sports, Science, and Technology (MEXT), Japan (Notification No.71). A complete overview of the protocol is shown in Figure 1.
1. Apparatus
<ol>
	<li>Prepare two kinds of cages: a single cage (width [W] &#215; depth [D] &#215; height [H] = 143 mm &#215; 293 mm &#215; 148 mm), and a &#8220;social defeat (SD)&#8221; cage (W &#215; D &#215; H = 220 mm &#215; 320 mm &#215; 135 mm).</li>
	<li>As shown in Figure 2, divide the SD cage into two compartments with an acrylic transparent board divider (5-mm thick) with 15 circular holes (3 &#215; 5 matrix: 8 mm in diameter).</li>
	<li>Obtain wood-shaving chips made from spruce trees, purified-diet food pellets, and a drinking water bottle. In addition, obtain paper towels, a mask, and latex gloves.</li>
	<li>For the social interaction test, prepare an open-field arena (W &#215; D &#215; H = 40 cm &#215; 40 cm &#215; 40 cm) made of gray polyvinylchloride, a weight made of steel (190 g), and a plastic interaction box (W &#215; D &#215; H = 10 cm &#215; 10 cm &#215; 13 cm; 100 g) with three wire-mesh windows (W &#215; D = 5 cm &#215; 5 cm) (Figure 3).</li>
	<li>Place a CCD camera (2.8&#8211;12 mm; F = 1:1.4; 1/3 inch CCD) and an automated tracking system in the behavioral testing room in the animal facility. Place an appropriate shelf for the mouse cages in the behavioral testing room to habituate the mice to the environment of the testing room for at least 30 min.</li>
</ol>
2. Habituation to the Environment
<ol>
	<li>Use male C57BL/6JJmsSlc (B6) mice that are 7 weeks old and male Slc:ICR (ICR) mice that are over 5 months old and deliver to the facility from an animal breeding company.</li>
	<li>Independently move two groups of B6 mice (n = 12 in each group) to the facility; use one group (screener B6 mice) for screening aggressive ICR mice and another group (subject B6 mice) for developing the sCSDS model.</li>
	<li>Introduce ICR mice (n = 12) to the facility in order to screen their aggressive behaviors.</li>
	<li>House the mice individually in single cages for 1 week under a 12-hr light-dark cycle (around 100 lux fluorescent light, lights on at 08:00) with constant temperature (around 23 &#176;C) and humidity levels (around 40%) in order to habituate them to the environment. Partition each cage by placing white-colored plastic boards between the cages so that the mice are not affected by the behaviors of neighboring mice.</li>
	<li>Make purified-diet food pellets and reverse osmosis water available ad libitum. Use AIN-93G chow because the ingredients of other non-purified diet pellets may vary.</li>
	<li>Measure body weight, food intake (FI), and water intake (WI) of the mice every day. Calculate the body weight gain (BWG) from the initial day.</li>
</ol>
3. Screening of Aggressive ICR Mice
<ol>
	<li>After habituation for 1 week as mentioned above, screen the ICR mice (resident) using resident-intruder tests for a 3 min trial with screener B6 mice (intruder; 8 weeks of age) in the afternoon (14:00-17:00) under lighting of around 300 lux in the housing room.
	<ol>
		<li>Specifically, test each ICR mouse for three trials per day for 5 consecutive days (15 trials in total) toward many different B6 mice as possible to find which ICR mice show high aggression toward the intruders. During the tests, record the attack latency and duration of the aggressive behavior (rapid motions with attack biting).</li>
	</ol>
	</li>
	<li>Identify hyper-aggressive ICR mice by checking the damage wounds on B6 intruder mice after each trial.</li>
	<li>Calculate the ratio of trials in which the attack latency is less than 30 sec as a first index of the aggression score.</li>
	<li>Calculate the ratio of trials in which the attack latency is less than 3 min as a second index of the aggression score.</li>
	<li>Evaluate the aggression scores from the first index. Use the second index when the first index is equal.</li>
	<li>Select the ICR mice that had high aggression scores without hyper-aggressive behaviors as aggressive ICR mice. Use the aggressive ICR mice repeatedly throughout the next set of experiments until approximately 12 months of age; however, perform the screening process for aggressive ICR mice as described above before every experiment to confirm the aggressiveness of ICR mice.</li>
	<li>After each session of the screening, record the degree of injury of the screener B6 mice. &#160;If a mouse is wounded,&#160;isolate the mouse in a single cage and observe the progress by checking its body weight gain, food intake, and water intake.&#160;In the case of severe wounds which affect physiology and behavior in mice, &#160;euthanize them according to local IACUC guidelines.</li>
</ol>
4. sCSDS
<ol>
	<li>Introduce subject B6 mice 7 days before (day -6) the day of the initial stress exposure (day 1) and house the mice individually in single cages to habituate them to the environment.</li>
	<li>Three days before day 1 (on day -2), move the aggressive ICR mice into a compartment of each SD cage, which is divided by an acrylic divider to let the mice establish their territories in the SD cages (the same number of subject B6 mice).</li>
	<li>Divide the SD cages by using white-colored partitioning boards, as in the single-cage condition described above.</li>
	<li>For non-stressed control B6 mice, prepare SD cages (half the number of mice) to keep the mice in pairs; place two mice into each compartment divided by the divider in an SD cage for 10 days.</li>
	<li>After the daily BWG, FI, and WI measurements on day 1, place subject B6 mice into one of the compartments of the SD cages (resident&#8217;s home-cage) in the afternoon (14:00&#8211;16:00) under lighting of around 300 lux in the housing room and measure the physical contact time from the first attack bite to complete the resident-intruder test.</li>
	<li>On day 1, set the physical contact time to 5 min from the first attack bite and have observers count the number of attack bites by ICR mice that are directed preferentially at the back or flanks of the opponent as described by Miczek et al.20 to determine the degree of physical stress to B6 mice.</li>
	<li>After the physical contact time, rescue subject B6 mice, and check and record their fur status and wounds. Then, place the B6 mice into another compartment next to the ICR mice in the SD cages until exposure to physical stress the next day.</li>
	<li>Because the acrylic divider in the SD cage is transparent and contains holes, expose subject B6 mice to various emotional stresses, including visual, auditory, and olfactory stimuli, from the ICR mice in the neighboring compartment of the SD cage for 24 hr every day.</li>
	<li>Measure the BWG, FI, and WI of control B6 mice daily and then place them into each compartment in the SD cages for 1 day.</li>
	<li>On day 2, after the daily measurements of BWG, FI, and WI, introduce subject B6 mice into the territories of other ICR mice to expose them to physical stress.</li>
	<li>Set the duration of physical contact at 4.5 min from the first attack bite on day 2, and count the number of attack bites.</li>
	<li>Move control B6 mice into different compartments and change the pair combinations in order to shuffle the placement and partner of the pairs every day.</li>
	<li>Decrease the duration of physical contact by 0.5 min per day, so that the duration on day 10 is set to 0.5 min from the first attack bite.</li>
	<li>On day 7, replace approximately half of the wood shavings in all of the compartments in the SD cages.</li>
	<li>After exposure to the last physical stress on day 10, move subject B6 mice into single cages. Similar to the subject B6 mice, move control B6 mice into single cages on day 10.</li>
	<li>If an ICR mouse does not show any attack bites until 5 min in each trial on days 1 to 10, terminate the trial. Replace the ICR mouse with spare aggressive ICR mice and conduct an alternative trial for the subject B6 mouse.</li>
	<li>If a mouse is wounded, isolate the mouse in a single cage and observe the progress by checking its body weight gain, food intake, and water intake. Follow your IACUC guidelines for analgesia.&#160;&#160;In the case of severe wounds which affect physiology and behavior in mice, euthanize them according to local IACUC guidelines.</li>
</ol>
5. Social Avoidance Test (Social Interaction Test)
<ol>
	<li>Perform behavioral tests for social avoidance in the morning (09:00&#8211;12:00) on day 11.</li>
	<li>Thirty minutes before the test, transfer the cages of control and subject B6 mice onto a shelf (dim light of less than 1 lux) in the behavioral testing room under lighting of less than 20 lux to allow them to habituate to the environment.</li>
	<li>To reduce order-effects, alternately test control and subject B6 mice.</li>
	<li>Clean an open-field arena (illuminated with a light of 20 lux at the center of the field) and a plastic interaction box using paper towels soaked with 70% ethanol before the behavioral test for each mouse to remove feces, urine, and any odors.</li>
	<li>Place an unfamiliar ICR mouse (not used as an aggressor) from a single cage near the open-field apparatus.</li>
	<li>Place a B6 mouse into the open field of the vacant interaction box as shown in Figure 4. Monitor and analyze its behavior for 2.5 min using the automatic analysis system (described in step 5.10).</li>
	<li>After the first trial, remove the B6 mouse from the field and place it into its home cage.</li>
	<li>Following this, place the unfamiliar male ICR mouse (social target) into the interaction box and then introduce the B6 mouse into the open field and monitor its behavior for 2.5 min.</li>
	<li>After the second trial, return both the B6 and ICR mice to their home cages, and clean the field and the interaction box as mentioned above.</li>
	<li>Repeat these steps for each B6 mouse to test its social behavior. During each trial, record top-view movies using a CCD camera.
	<ol>
		<li>After the behavioral tests, calculate the time spent in the interaction zone (in seconds) and in the corner zone (in seconds) for each trial as shown in Goto et al.13. Judge the position of the mouse based on the center of the mouse.</li>
		<li>Calculate the social interaction scores as 100 &#215; (interaction time, with target)/(interaction time, without target), following the methods published in Krishnan et al.21.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Heim, C., Binder, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+research+trends+in+early+life+stress+and+depression:+Review+of+human+studies+on+sensitive+periods,+gene-environment+interactions,+and+epigenetics.">Current research trends in early life stress and depression: Review of human studies on sensitive periods, gene-environment interactions, and epigenetics.</a> Exp. Neurol. 233 (1), 102-111 (2012).</li><li>Whiteford, H. 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=Global+burden+of+disease+attributable+to+mental+and+substance+use+disorders:+findings+from+the+Global+Burden+of+Disease+Study+2010.">Global burden of disease attributable to mental and substance use disorders: findings from the Global Burden of Disease Study 2010.</a> Lancet. 382 (9904), 1575-1586 (2013).</li><li>Ferrari, A. 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=The+Burden+Attributable+to+Mental+and+Substance+Use+Disorders+as+Risk+Factors+for+Suicide:+Findings+from+the+Global+Burden+of+Disease+Study+2010.">The Burden Attributable to Mental and Substance Use Disorders as Risk Factors for Suicide: Findings from the Global Burden of Disease Study 2010.</a> PLoS ONE. 9 (4), e91936 (2014).</li><li>Flint, J., Kendler, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+major+depression.">The genetics of major depression.</a> Neuron. 81 (3), 484-503 (2014).</li><li>Nestler, E. 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=Neurobiology+of+Depression.">Neurobiology of Depression.</a> Neuron. 34 (1), 13-25 (2002).</li><li>Gold, P. W., Chrousos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+stress+system+and+its+dysregulation+in+melancholic+and+atypical+depression:+high+vs+low+CRH/NE+states.">Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states.</a> Mol. Psychiatry. 7 (3), 254-275 (2002).</li><li>O'Keane, V., Frodl, T., Dinan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+atypical+depression+in+relation+to+the+course+of+depression+and+changes+in+HPA+axis+organization.">A review of atypical depression in relation to the course of depression and changes in HPA axis organization.</a> Psychoneuroendocrinology. 37 (10), 1589-1599 (2012).</li><li>Nestler, E. J., 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=Animal+models+of+neuropsychiatric+disorders.">Animal models of neuropsychiatric disorders.</a> Nat. Neurosci. 13 (10), 1161-1169 (2010).</li><li>Katz, 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=Animal+models+and+human+depressive+disorders.">Animal models and human depressive disorders.</a> Neurosci. Biobehav. Rev. 5 (2), 231-246 (1981).</li><li>Krishnan, V., Nestler, E. J. <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+depression:+molecular+perspectives.">Animal models of depression: molecular perspectives.</a> Curr. Top. Behav. Neurosci. 7, 121-147 (2011).</li><li>Kudryavtseva, N. N., Bakshtanovskaya, I. V., Koryakina, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+model+of+depression+in+mice+of+C57BL/6J+strain.">Social model of depression in mice of C57BL/6J strain.</a> Pharmacol. Biochem. Behav. 38 (2), 315-320 (1991).</li><li>Miczek, K. A., Yap, J. J., Covington, H. 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+stress,+therapeutics+and+drug+abuse:+preclinical+models+of+escalated+and+depressed+intake.">Social stress, therapeutics and drug abuse: preclinical models of escalated and depressed intake.</a> Pharmacol. Ther. 120 (2), 102-128 (2008).</li><li>Goto, 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=Subchronic+and+mild+social+defeat+stress+accelerates+food+intake+and+body+weight+gain+with+polydipsia-like+features+in+mice.">Subchronic and mild social defeat stress accelerates food intake and body weight gain with polydipsia-like features in mice.</a> Behav. Brain Res. 270, 339-348 (2014).</li><li>Goto, T., Kubota, Y., Toyoda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+Liver+Metabolic+Profiles+in+Mice+Subjected+to+Subchronic+and+Mild+Social+Defeat+Stress.">Plasma and Liver Metabolic Profiles in Mice Subjected to Subchronic and Mild Social Defeat Stress.</a> J. Proteome Res. , (2014).</li><li>Iio, W., Matsukawa, N., Tsukahara, T., Kohari, D., Toyoda, A. <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+chronic+social+defeat+stress+on+MAP+kinase+cascade.">Effects of chronic social defeat stress on MAP kinase cascade.</a> Neurosci. Lett. 504 (3), 281-284 (2011).</li><li>Iio, W., 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=Effects+of+chronic+social+defeat+stress+on+peripheral+leptin+and+its+hypothalamic+actions.">Effects of chronic social defeat stress on peripheral leptin and its hypothalamic actions.</a> BMC Neurosci. 15 (72), (2014).</li><li>Iio, W., 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=Anorexic+behavior+and+elevation+of+hypothalamic+malonyl-CoA+in+socially+defeated+rats.">Anorexic behavior and elevation of hypothalamic malonyl-CoA in socially defeated rats.</a> Biochem. Biophys. Res. Commun. 421 (2), 301-304 (2012).</li><li>Crawley, J. 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> What's Wrong With My Mouse?: Behavioral Phenotyping of Transgenic and Knockout Mice. , 978-970 (2007).</li><li>Golden, S. A., Covington, H. E., Berton, O., Russo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+repeated+social+defeat+stress+in+mice.">A standardized protocol for repeated social defeat stress in mice.</a> Nat. Protoc. 6 (8), 1183-1191 (2011).</li><li>Miczek, K. A., Maxson, S. C., Fish, E. W., Faccidomo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggressive+behavioral+phenotypes+in+mice.">Aggressive behavioral phenotypes in mice.</a> Behav. Brain Res. 125, 167-181 (2001).</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>Chuang, J. 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+beta3-adrenergic-leptin-melanocortin+circuit+regulates+behavioral+and+metabolic+changes+induced+by+chronic+stress.">A beta3-adrenergic-leptin-melanocortin circuit regulates behavioral and metabolic changes induced by chronic stress.</a> Biol. Psychiatry. 67 (11), 1075-1082 (2010).</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> Biol. Psychiatry. 73 (1), 7-14 (2013).</li><li>Savignac, H. 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=Increased+sensitivity+to+the+effects+of+chronic+social+defeat+stress+in+an+innately+anxious+mouse+strain.">Increased sensitivity to the effects of chronic social defeat stress in an innately anxious mouse strain.</a> Neuroscience. 192, 524-536 (2011).</li><li>Green, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+Adversities+and+Adult+Psychiatric+Disorders+in+the+National+Comorbidity+Survey+Replication+I:+associations+with+first+onset+of+DSM-IV+disorders.">Childhood Adversities and Adult Psychiatric Disorders in the National Comorbidity Survey Replication I: associations with first onset of DSM-IV disorders.</a> Arch. Gen. Psychiatry. 67 (2), 113-123 (2010).</li><li>Miniati, 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=Clinical+characteristics+and+treatment+outcome+of+depression+in+patients+with+and+without+a+history+of+emotional+and+physical+abuse.">Clinical characteristics and treatment outcome of depression in patients with and without a history of emotional and physical abuse.</a> J. Psychiatr. Res. 44 (5), 302-309 (2010).</li><li>Tao, 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=Examining+the+relationship+between+lifetime+stressful+life+events+and+the+onset+of+major+depression+in+Chinese+women.">Examining the relationship between lifetime stressful life events and the onset of major depression in Chinese women.</a> J. Affect. Disord. 135 (1-3), 95-99 (2011).</li><li>Valente, S., Fisher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognizing+and+managing+psychogenic+polydipsia+in+mental+health.">Recognizing and managing psychogenic polydipsia in mental health.</a> J. Nurse Pract. 6 (7), 546-550 (2010).</li><li>Dundas, B., Harris, M., Narasimham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychogenic+Polydipsia+Review:+Etiology,+Differential,+and+Treatment.">Psychogenic Polydipsia Review: Etiology, Differential, and Treatment.</a> Curr. Psychiatry Rep. 9 (3), 236-241 (2007).</li><li>Tsumura, K., 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=Downregulation+of+AQP2+expression+in+the+kidney+of+polydipsic+STR/N+mice.">Downregulation of AQP2 expression in the kidney of polydipsic STR/N mice.</a> Am. J. Physiol. Renal Physiol. 290 (2), F478-F485 (2006).</li><li>Flores, 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=Schedule-Induced+Polydipsia:+Searching+for+the+Endophenotype+of+Compulsive+Behavior.">Schedule-Induced Polydipsia: Searching for the Endophenotype of Compulsive Behavior.</a> World J. Neurosci. 4, 253-260 (2014).</li><li>Cryan, J. F., Dinan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind-altering+microorganisms:+the+impact+of+the+gut+microbiota+on+brain+and+and+behaviour.">Mind-altering microorganisms: the impact of the gut microbiota on brain and and behaviour.</a> Nat. Rev. Neurosci. 13 (10), 701-712 (2012).</li></ol>

The authors declare that they have no competing financial interests.

There were definitive differences in body weight between sCSDS mice and CSDS mice subjected to the standard CSDS protocol (5 to 10 min of physical contact with aggressors per day)19. The sCSDS mice showed increased BWG during the stress period, whereas the standard CSDS mice showed a decrease in body weight during the stress period21,22,23. There is a substantial difference between the two protocols in terms of the total duration of physical contact with aggressors during the 10-day stress period. W...

Many kinds of stressful events occur throughout the lives of humans. Excessive stress often leads to harmful physiological consequences in humans and animals. In humans, stressful events are major risk factors for precipitating psychiatric disorders such as depression1. A Global Burden of Disease study indicated that depression is one of the most disabling disorders in terms of disability-adjusted life years (DALYs) and years lived with disability2. In addition, depression accounts for the largest proportion of suicide DALYs3. People suffering from depression find it difficult to manage their lives, and as a result, their quality of life often worsens. Therefore, there is a strong need to develop effective therapeutics to improve the quality of life in these patients.
Many studies have been performed on major depressive disorders, and have revealed that the genetic contribution to disease susceptibility is approximately 30&#8211;40%, which is explained by the effects of multiple loci of small effects4. Because of the complex pathogenic mechanisms underlying depression, the detailed etiology of the disease remains elusive. Clinical reports indicate that there are subtypes of depression such as melancholic and atypical depression5, which show reduced and increased body weight, respectively6. Although 25&#8211;30% and 15&#8211;30% of patients with depression represent pure melancholic and atypical features, respectively, most of them have mixed features of both subtypes7. Therefore, major depression has a wide range of symptoms. In order to find biomarkers and develop objective therapeutics for the various types of depression in humans, it is important to establish several different animal models of depression8.
Animal models of depression have been established using several approaches including learned helplessness, chronic unpredictable mild stress, and chronic social defeat stress (CSDS)9-12. Toyoda and colleagues established the CSDS models of rats and mice13-17 in order to elucidate the metabolism and behaviors that are associated with depression. Given that animal models of depression are evaluated by face validity18, the context within which the model is established is important. Moreover, Golden et al.19 reported the methods for creating CSDS mice in detail. It is known that the deficits in social behavior of CSDS mice can be recovered by chronic treatment, but not by acute treatment, with antidepressants, and that they share symptoms similar to those in patients with depression in terms of the regulation of brain-derived neurotrophic factor6.
Goto et al.13 previously developed the subchronic and mild social defeat stress (sCSDS) mouse model by modifying the methods of Golden et al.19. The sCSDS mice show polydipsia- and hyperphagia-like symptoms following gains in body weight and increased body water content13. In this report, the protocol for establishing the sCSDS mouse model is provided and we discuss the utility of this model.

<table><tbody><tr><td>single cage</td><td>Charles River Laboratories Japan</td><td></td><td>width [W] &amp;times; depth [D] &amp;times; height [H] = 143 &amp;times; 293 &amp;times; 148 mm</td></tr><tr><td>M cage</td><td>Natsume Seisakusho</td><td></td><td>W &amp;times; D &amp;times; H = 220 &amp;times; 320 &amp;times; 135 mm</td></tr><tr><td>Whiteflake</td><td>Charles River Laboratories Japan</td><td></td><td>Wood-shaving chips made from spruce trees</td></tr><tr><td>AIN-93G</td><td>Oriental Yeast</td><td></td><td>purified-diet food pellets</td></tr><tr><td>Kimtowel</td><td>Nippon Paper Crecia Co.</td><td></td><td>Paper towels</td></tr><tr><td>open-field arena</td><td>O&amp;rsquo;Hara &amp;amp; Co.</td><td></td><td>made of gray polyvinylchloride</td></tr></tbody></table>

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.

To monitor the degree of physical stress for over the 10-day periods, the number of attack bites by ICR mice was manually counted by a researcher. Figure 5A indicates the individual values for the number of attack bites received. There was considerable variability in the early stage (approximately 10&#8211;120 bites on day 1), but this variability was reduced in the later stage (approximately 5&#8211;20 bites on day 10). Figure 5B&#160;indicates that the average number of attack bites re...

Watch this Scientific Journal Video about A Mouse Model of Subchronic and Mild Social Defeat Stress for Understanding Stress-induced Behavioral and Physiological Deficits at JoVE.com

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

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 ...

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Research

JoVE Journal

Behavior

19.6K Views.  Ibaraki University. 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.

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

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

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

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

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

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

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

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

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

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...

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

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.

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

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

Chronic Social Defeat Stress in Early Adolescent Male Mice

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

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

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

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression,&#160;as well as decreased cognition.

Presented here is a social isolation (SI)-induced anxiety mouse model that utilizes wild type C56BL/6J mice to induce stress and anxiety-like behavior with minimal handling and no invasive procedures. This model reflects modern life patterns of social isolation and is ideal for studying anxiety and related disorders.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of ...

Neuroscience

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological ...

Medicine

Modeling Social Stress

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are unfamiliar with or have difficulty adjusting to. Since it is hard to examine mechanisms of social stress in humans, modeling this condition in animals may help scientist in developing new therapies for treating this commonly encountered problem.
This science education video begins by discussing the known anatomy and physiology behind stress response. Then, we explain a well-established paradigm for modeling social stress in rodents, the Resident-Intruder task. In the applications section, we review some example studies in which response to stress is measured.

Physiology of Stress; Social Stress- Resident-Intruder Task

Stress negatively affects our quality of life. In particular, some individuals experience social stress when placed in a social environment that they are ...