This work was funded by the Canadian Institutes for Health Research (CF Grant Numbers: MOP-74709; PJT 190045), the National Institute on Drug Abuse (CF Grant number: R01DA037911), the Natural Sciences and Engineering Research Council of Canada (CF Grant Number: 2982226). Andrea Pantoja-Urban was supported by The National Council for Humanities, Science and Technologies/Consejo Nacional de Humanidades, Ciencias y Tecnolog&#237;as (CONAHCYT) from M&#233;xico and FRQNT - Merit scholarship program for foreign students (PBEEE). Samuel Richer was supported by a scholarship from the Integrated Program of Neuroscience at McGill University. Figure illustrations were created using templates from BioRender.com.

Experimental procedures were performed in accordance with the guidelines of the Canadian Council of Animal Care and approved by the McGill University and Douglas Hospital Animal Care Committee (animal experiment approval number: 2005-5084). All mice were housed in a temperature- and humidity-controlled (21-22 &#176;C; 60%) colony room and on a 12 h light-dark cycle (light on at 8:00 h) at the Neurophenotyping Centre of the Douglas Mental Health University Institute. The mice had ad libitum access to food and water throughout the experiments. Mice were assigned randomly to each experimental condition. Experimental animals were obtained from a commercial source (see Table of Materials). Table 2 summarizes the animal&#39;s characteristics for each experimental phase. In total, 489 adolescent C57BL/6J mice (259 males; 230 females) were used across 20 experimental cohorts.
Table 2: Animal subjects for each experimental phase.&#160;&#8224;If these animals are not severely injured, they can be reused for priming. &#8225;If these animals are not severely injured, they can be reused for priming during AcSD, &#167;Neurophenotyping Centre of the Douglas Mental Health University Institute. &#182;For experiments with males, #For experiments with females, if defeat is mixed, use females for priming. &#8224;&#8224;Not to be used for AcSD. Abbreviations: AcSD = accelerated social defeat stress; PND = Postnatal day <a href="https://www.jove.com/files/ftp_upload/66455/Table 2_RE.xlsx" target="_blank">Please click here to download this Table.</a>
1. Acclimation
NOTE: This phase should last 1 week minimum.
<ol>
	<li>Purchase male CD-1 retired breeder mice (see Table of Materials) older than 4 months of age and twice the quantity of the adolescent C57BL/6J mice (i.e., if 10 mice will be exposed to stress, purchase 20 CD-1 mice). 
	NOTE: Acquiring this quantity of CD-1 mice is recommended because just a small proportion of CD-1 mice show aggressive behavior towards adolescent mice.</li>
	<li>Let the CD-1 male mice habituate to the new facility in their home cage with food and water ad libitum for 1 week prior to the screening and priming.</li>
	<li>Use C57BL/6 at PND &#62;65 (adults) and PND 25 &#177; 3 (adolescents) (Table 2) for the screening and priming sessions. Acquire 4 C57BL/6 mice of each age per day of priming (i.e., for 3 days of priming, acquire 12 PND &#62;65 and 12 PND 25 &#177; 3) to decrease the risk of injuries because these animals will be employed repeatedly for screening and priming process.</li>
</ol>
2. CD-1 screening and priming
NOTE: The duration of this phase is 3 days of screening (1 day for female mice) and 3-4 days of priming (this can increase for female mice and depending on the CD-1 mice aggressive behavior.)
<ol>
	<li>Use the operational definition of attack to register the attacks of the CD-1 as described below.
	<ol>
		<li>One attack is defined as when the CD-1 mouse bites the C57BL/6J mouse, and the C57BL/6J mouse moves away in response to the bite. A bite is defined as when the CD-1 mouse places its teeth on any part of the C57BL/6J mouse&#39;s body. Moving away is defined as when the C57BL/6J mouse moves both of its hind paws from the position they were in before the CD-1 engaged it.</li>
		<li>Ensure there is a difference of at least ~2 s between separate attacks. If more than one bite occurs &#60;2 s apart, count as one attack.</li>
		<li>If the CD-1 mouse bites more than once in succession without a break (&#60;~2 s apart), gently separate animals with a plastic ruler. If the CD-1 mouse bites and does not let go, gently separate the animals. If the C57BL/6J mouse becomes trapped in a corner or is pinned down by the CD-1 mouse and cannot move away in response to a bite, gently separate animals with a ruler. Count all of these as one attack (up to separation).</li>
	</ol>
	</li>
	<li>Screening
	<ol>
		<li>Use nitrile examination gloves for all animal handling and avoid the use of perfume or fragrances.</li>
		<li>Perform the 1st day of the screening only with adult C57BL/6 male mice as intruders (PND &#62;65) in the CD-1 mice home-cage. Always screen the CD-1 mice before a new experiment.</li>
		<li>Introduce an adult male C57BL/6 mouse PND &#62;65 in the home-cage of a CD-1 male mouse for 3 min. Register the latency to the first aggression and how many aggressions are performed (see Supplementary Table 1).</li>
		<li>From the 2nd to the last day of screening, introduce an adult male C57BL/6 PND&#62;65 for 1 min (limit to 30 s if the CD-1 is very aggressive) and then swap it with an adolescent C57BL/6 PND 25 &#177; 3 for 5 min. 
		NOTE: It is important to perform the screening with adolescent mice because not all the mice that attack the adult C57BL/6 male mouse will attack the adolescent C57BL/6. In fact, CD-1 mice that attack adolescent mice will not always attack adult C57BL/6 males.</li>
		<li>If the experiment&#160;is for aggressiveness towards female mice, perform the screening and priming with adolescent female mice, even if the experimental mice include both sexes.</li>
		<li>Register the latency to the first aggression, the number of attacks, and the wounds received by all the C57BL/6 mice (see Supplementary Table 2).</li>
		<li>Register any kind of interaction: chasing, mounting behaviors, sniffing, grooming, or biting. Register if the CD-1 does not approach the adolescent C57BL/6 mice; if this behavior is repeated for 2 days, do not include the CD-1 in priming.</li>
		<li>For the priming phase, select all the CD-1 mice that display behaviors such as chasing, mounting behaviors, sniffing, following, grooming, or biting the adolescent C57BL/6.</li>
		<li>Keep the non-selected CD-1 mice and use them for the social interaction test (SIT). 
		NOTE: Territorial behavior increases with time; therefore, the non-selected CD-1 mice may become aggressive toward adolescent mice in the next experiment.</li>
	</ol>
	</li>
	<li>Priming CD-1 aggressive behavior
	<ol>
		<li>House the CD-1 mice selected for priming in their home cage until the last day of priming.</li>
		<li>Introduce an adult male C57BL/6J mouse PND &#62;65 for 30 s to the CD-1 home-cage and then swap it with an adolescent C57BL/6 PND 25 &#177; 3 mouse for 5 min.</li>
		<li>Register the latency to the first aggression, the number of attacks, and any interaction (see Supplementary Table 3).</li>
		<li>Perform the priming 2x a day for 3-4 days at the same time the defeats are scheduled (9:00 am and 2:00 pm). 
		NOTE: Not all the CD-1 mice will attack the adolescent mice during the first days, especially if the priming is towards female mice. The number of days for priming can increase or decrease depending on the aggressive behavior shown by the CD-1 mice.</li>
		<li>To decrease the number of priming days, reuse CD-1 mice that have been through a prior social defeat experiment with male mice. If CD-1 mice do not show increased aggression, extend the days for priming until reaching criteria.</li>
		<li>Decrease the days for priming if the CD-1 mice show extreme aggressive behavior (C57BL/6 mice show excessive wounding) to prevent severe injuries during social defeat.</li>
		<li>For the next phase of the experiment, select the CD-1 mice that attack the adolescent mice at least 10x (males) or 5x (females) for 2 consecutive days.</li>
	</ol>
	</li>
</ol>
3. AcSD protocol
NOTE: This phase consists of 3 days of habituation and 4 days of exposure to AcSD.
<ol>
	<li>Habituation
	<ol>
		<li>Set-up the rat cages for social defeat (Figure 1A-F). Divide the rat cages into 2 compartments using appropriate acrylic glass dividers (Figure 1B,D; see Table of Materials).</li>
		<li>Add hard woodchip bedding to both sides of the cage, add a cotton square per side as bedding material (Figure 1D). Ensure that the wire top is secured with binder clips (Figure 1E). Ensure that the rat cage is covered with the cover lid without the protective cotton (Figure 1F).</li>
		<li>Place a CD-1 mouse per cage on one side of the cage. Place a tag on the outside of the cage for easy identification of each mouse.</li>
		<li>House the selected CD-1 mice in the social defeat apparatus (Figure 1F) 2 or 3 days before the AcSD protocol starts (to enhance CD-1 territorial behavior).</li>
		<li>Perform a priming session 1 day before the experiment; this is important to keep aggression consistent.</li>
	</ol>
	</li>
	<li>Defeat sessions
	<ol>
		<li>Perform 2 sessions per day (first session: 9 AM, second session: 2 PM) for 4 days, 8 sessions in total. Make a schedule to ensure that the aggressor will be different for each session (see Supplementary Table 4 and Supplementary Table 5).</li>
		<li>Weigh the C57BL/6 adolescent mouse before introducing it to the CD-1 side of the cage. Introduce an adult male C57BL/6 mouse &#62;65 for 30 s into the CD-1 side of the defeat cage and then swap it with an adolescent C57BL/6 PND 24-28 mouse for a 10-min long defeat episode or 10 attacks maximum per session.</li>
		<li>Separate the animals after each attack with a plastic ruler. After every session of defeat, check for injuries, and treat them with pain relief cream. Register the number of injuries.</li>
		<li>Place the adolescent C57BL/6J mouse in the free compartment next to an unfamiliar aggressor until the next session. Repeat the procedure with a different aggressor each session.</li>
		<li>Start the session with the experimental and aggressor mice that were not paired the previous session. Monitor the injuries of the adult C57BL/6J male mice used for priming (see Supplementary Table 6).</li>
	</ol>
	</li>
	<li>Follow the recommendations below for animal welfare and humane endpoints.
	<ol>
		<li>Follow the standard operating procedure of the corresponding institutional review board for social defeats and ensure constant veterinary evaluation.</li>
		<li>Monitor the health of mice throughout the study to avoid animal suffering and death.</li>
		<li>Verify that the perforated divider is correctly set to prevent mice from crossing to the other compartment.</li>
		<li>Notify the veterinary care staff when an animal has a wound for appropriate monitoring and apply pain relief cream daily until the wound is completely healed.</li>
		<li>If an injury worsens (wounds increase in size), limit the attacks to 5 in the next session allowing recovery. Check for severe injuries. Severe injuries are defined as wounds totaling &#62; 1 cm.</li>
		<li>Immediately remove and euthanize animals presenting a 15%-20% reduction of their weight from initial body weight; reduced/decreased locomotor activity and impaired grooming; mice with wounds &#62; 1 cm; severed penis, or bite marks with swelling of the forelimbs. 
		NOTE: Primary euthanasia is conducted via isoflurane/CO2 gas chambers followed by secondary euthanasia done by cervical dislocation.</li>
	</ol>
	</li>
	<li>Control mice
	<ol>
		<li>Perform 2 sessions per day (first session: 9 AM, second session: 2 PM) for 4 days, 8 sessions in total. Make a schedule to ensure that control mice share the cage with a different C57BL/6J&#160;conspecific every session (see Supplementary Table 7 and Supplementary Table 8). 
		NOTE: Control mice are not exposed to an aggressor. To mimic the introduction to a novel CD-1 mouse every defeat session, control mice are paired with a new same-sex and same-age conspecific (non-littermate) every session.</li>
		<li>House the control mice in the social defeat apparatus (Figure 1) in a separate room from that of the AcSD mice.</li>
		<li>Place two control mice per cage, one on each side of the cage. House the mice in a different side of the cage or different cage altogether, so they do not recognize the odor. Rotate the control mice each session.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66455/66455fig01.jpg" /> 
Figure 1: Apparatus for the accelerated social defeat stress (AcSD) for adolescent mice. (A) From left to right, paired steel-wire tops with 0.6 cm of separation between bars, clear perforated polycarbonate divider with perforations diameter of 0.6 cm, binder clips to secure the wire tops, clear rat cage with cover lid (without cotton protective layer). (B) Up: comparison between the polycarbonate dividers for chronic social defeat in adults (left) and AcSD in adolescents (right); down: close up to the polycarbonate divider for adolescent mice. (C) Up: comparison between the steel-wire tops for chronic social defeat in adults (left) and AcSD in adolescents (right); down: close-up of the steel-wire tops. (D-F) Assembling of the social defeat apparatus. <a href="https://www.jove.com/files/ftp_upload/66455/66455fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Social interaction test
<ol>
	<li>Perform the SIT following the AcSD as described previously57,58,59.
	<ol>
		<li>Perform the SIT 24 h after the last session of AcSD. Perform the SIT under red light conditions (not under yellow light conditions) between 9 AM and 4 PM.</li>
		<li>Set up the main squared-arena and the overhead video camera (see Table of Materials). The main arena is divided into 3 areas: the social interaction zone (SIZ), the corners, and the center; these areas should be marked in the arena with a black line (use a black permanent marker). Use the following dimensions and criteria for each area.
		<ol>
			<li>Ensure the SIZ is the squared area 14 cm x 9 cm, that surrounds the enclosure. The corners are small, squared areas (9 cm x 9 cm) in the corners of the wall opposite to the enclosure and represent the farthest point from the SIZ. The center is the area between the SIZ and the corners.</li>
		</ol>
		</li>
		<li>Clean the arena and the enclosures with a cleaning hydrogen peroxidase solution.</li>
		<li>Place identification tags for each animal on the floor for analysis. Record all animal behavior for offline analysis.</li>
	</ol>
	</li>
	<li>First session to determine baseline exploration
	<ol>
		<li>Place an empty wire mesh enclosure against one of the walls of the arena. Place the&#160;experimental (defeated or control) adolescent C57BL/6 mouse in the middle of the arena. Allow it to explore the arena freely for 2.5 min. Retrieve the mouse.</li>
		<li>Clean the arena and the enclosures with a cleaning hydrogen peroxidase solution.</li>
	</ol>
	</li>
	<li>Second session for social interaction
	<ol>
		<li>Place an unfamiliar CD-1 mouse inside the wire mesh enclosure. Place the enclosure in the same location as the previous session.</li>
		<li>Reintroduce the same adolescent C57BL/6 mouse to the arena. Allow it to explore the arena freely for 2.5 min. Retrieve the mice.</li>
		<li>Clean the arena and the enclosures with a cleaning hydrogen peroxidase solution.</li>
		<li>Repeat the process for all adolescent C57BL/6 mice (stressed and control mice) in a counterbalanced order.</li>
		<li>Use more than one CD-1 mouse for the SIT to decrease handling stress and bias (e.g., for a cohort of 30 C57BL/6 mice, use 4 CD-1 mice). Counterbalance them among the adolescent mice. 
		NOTE: This test measures the approach and/or avoidance behavior of the experimental mice toward a social target, as first described by Krishnan5 and Golden7.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>Record the time (s) spent in the interaction zone and the corners during both sessions of the test.</li>
		<li>Calculate the social interaction ratio as the time spent in the interaction zone with the CD-1 aggressor present divided by the time spent in the interaction zone with the CD-1 aggressor absent.</li>
		<li>Classify stressed mice with a ratio &#60; 1.00 as susceptible and a ratio &#8805; 1.00 as resilient.</li>
		<li>Exclude animals that do not explore the arena during the first session (i.e., spent 0 s in any of the designated areas) from all analyses to control differences in locomotor activity.</li>
		<li>Exclude all animals that receive 0 attacks in &#8805;4 social defeat sessions from all analyses to prevent non-representative resilient animals.</li>
		<li>Exclude mice (not susceptible) that do not spend at least 61 s inside the interaction zone to ensure that high social interaction ratios reflect actual interest in the social target.</li>
		<li>Exclude mice with outlier interaction ratio scores.</li>
	</ol>
	</li>
</ol>

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

The authors have nothing to disclose.

Consistent, aggressive behavior in CD-1 mice 
During the screening phase, it is very important to take note of all the behaviors displayed by the CD-1 (chasing, mounting behaviors, sniffing, grooming, or biting) and to closely follow these records when selecting the CD-1 mice for the AcSD. It is likely that a CD-1 mouse which interacts with the adolescent mouse without attacking it will develop aggressiveness towards adolescent mice during priming. In contrast, a CD-1 mouse which&#160;attacks an adu...

The chronic social defeat stress paradigm is widely used to model social stress in adult postnatal day (PND) &#62;65 male rodents. This paradigm is based on the natural aggressive behavior of an adult male rodent when an intruder invades its territory. This model is used in a variety of rodent species, including rats, hamsters, and mice1,2,3,4,5,6,7,8,9, and consists of a combination of physical aggression and psychological stress lasting about 10 days, during which an intruder rodent experiments a few min of physical aggression from the resident rodent. The two rodents remain in the resident&#39;s home cage, separated by a divider that allows sensory but not physical contact7. In mice experiments, the most commonly used resident/aggressor mice are retired breeders Swiss CD-1 mice, which show robust territorial behavior against intruder mice6,7. For the intruder mice, the best characterized strain is the inbreed C57BL/6 strain2,4,5. The defeated mouse is exposed to a new aggressor every day to prevent habituation to the aggressor. Control mice are housed with a different conspecific each day. At 24 h after the last defeat session, experimental mice are tested in a social interaction test (SIT) in which they can explore an open field in the absence (no target), or presence of a novel CD-1 mouse (target). Control mice spend more time in the interaction zone with the target versus the non-target part of the task. Defeated mice are classified as susceptible (ratio&#60;1) or resilient (ratio&#62;1) according to a social interaction ratio (time spent in the interaction zone with aggressor present/time spent in the interaction zone with aggressor absent). This procedure provides a useful tool to study individual differences in response to stress.
Until recent years, the chronic social defeat stress model has been used mostly in adult male mice because the accentuated male dominance hierarchies involve fighting against males but not against females6,7. Furthermore, male rodents typically do not attack females; instead, they engage in mating behaviors10. Notwithstanding these obstacles, different strategies have been developed to adapt the chronic social defeat stress paradigm for adult female mice. For example, the California mouse model of social defeat is based on the natural aggression of this monogamous species from both sexes when defending their territory9,11. Other approaches focus on inducing aggressive behavior of the CD-1 mice by stimulating their ventromedial hypothalamus to have consistent aggressive behavior10,12, or by applying male urine in the experimental adult female mice to receive attacks from CD-1 aggressors13. This heightened and consistent aggression of the CD-1 mice is critical for the experimental intruder mouse to show clear behavioral signs of subordination towards the repeated attacks by the aggressor during length of the interaction6.
Adapting the chronic social defeat model to use in adolescent C57BL/6mice 
Adolescence is a period marked by substantial psychosocial maturation, which unfolds in parallel with changes in the micro and macro architecture of the brain, particularly in the prefrontal cortex. In both humans and rodents, there is little consensus regarding the specific onset and end of the adolescent period14,15. Furthermore, there are critical windows of vulnerability within adolescence for experience-induced disruption of ongoing brain and cognitive development16,17,18,19. Puberty and adolescence occur at the same time, but these terms are not synonymous. Puberty signifies the onset of sexual maturation, whereas adolescence represents a broader phase characterized by the gradual shift from a juvenile state to achieving independence20. Different groups have suggested that adolescence in mice spans from weaning (PND 21) until adulthood (PND 60)21. Particularly, early adolescence can be referred to as the first and second weeks postweaning (PND 21-34) and mid-adolescence as the PND 35-48 period. These ranges encompass discrete developmental periods regarding for example the development of the dopamine system22,23,24, vulnerability to drug effects on developing neuronal networks17,25,26,27, and distinct behavioral characteristics16,20,28,29,30.
Fighting behavior from the resident mouse is required for the social defeat protocol. However, as with female mice, males do not typically engage in aggressive interactions with early adolescent mice, possibly because they do not perceive them as a threat. Most studies exploring the effects of chronic social defeat in adolescent C57BL/6 mice have been performed during the mid-adolescent period31,32,33,34,35; others do not specify the postnatal day of adolescent exposure36,37, or extend the days for the defeat to early adulthood38 or do not allow sensory contact39; other studies on adolescent mice use different strains40,41. The characteristics of these studies using chronic social defeat stress in adolescent male mice are summarized in Table 1.
Our research group is interested in targeting specific adolescent windows of exposure, including early adolescence, in C57BL/6 mice. Because of the short duration of the different adolescent periods, a modified version of the accelerated version of the chronic social defeat stress paradigm was designed42. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Previous work shows that there are significant sex differences in sensitivity to social stress in adolescence in rats8,26,43,44,45, as well as in the harmful effects of social stress on mental health trajectories in humans46,47,48,49,50,51,52,53,54,55,56. The AcSD model works effectively for adolescent female mice, too, allowing them to investigate potential sex-specific consequences as well as explore the neurobiological foundation.
Table 1: Studies using social defeat stress paradigms in adolescent male mice. Strain and species: California Mouse: Peromyscus californicus. C57BL/6: Mus musculus black 6 inbred mouse strain. C57BL/6J: M. musculus black 6 inbred mouse model provided by Jackson&#39;s laboratories. CD-1: M. musculus from Swiss outbred albino mouse strain. ICR: M. musculus Institute of Cancer Research outbred albino mouse strain. OF1: M. musculus Oncins France 1 outbred albino mouse strain. 
Abbreviations: wk = week; PND = Postnatal day; res = resilient; sus = susceptible; unsus = unsusceptible. <a href="https://www.jove.com/files/ftp_upload/66455/Table 1_RE.xlsx" target="_blank">Please click here to download this Table.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6 adolescent mice&#160;</td>
			<td>In house breeding</td>
			<td></td>
			<td>Mice were breeded at the Neurophenotyping Centre of the Douglas Mental Health University Institute.</td>
		</tr>
		<tr>
			<td>C57BL/6 adult mice&#160;</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code: 027</td>
			<td>Mice are ordered so as to arrive at PND&#62;65 and are group housed (four mice per cage) in standard mice cages.</td>
		</tr>
		<tr>
			<td>C57BL/6J adolescent mice&#160;</td>
			<td>Jackson Labs</td>
			<td>Strain Code: 000664; RRID:IMSR_JAX:000664</td>
			<td>Mice are ordered so as to arrive at PND 24 and are group housed (four mice per cage) in standard mice cages.</td>
		</tr>
		<tr>
			<td>CD-1 mice&#160;</td>
			<td>Charles River Laboratories</td>
			<td>Strain Code: 022</td>
			<td>Mice retired breeders more than three months of age and singled housed throughout.</td>
		</tr>
		<tr>
			<td>Cleaning solution&#160;</td>
			<td>Virox Animal Health</td>
			<td>DIN 02537222</td>
			<td>Prevail: Accelerated Hydrogen Peroxide. Desinfectant cleaner and deodorizer.</td>
		</tr>
		<tr>
			<td>Clear perforated acrylic glass divider&#160;</td>
			<td>Manufactured by Douglas Hospital, custom order</td>
			<td></td>
			<td>0.6 cm (w) &#215; 45.7 cm (d) &#215; 22.23 cm (h); perforations of 0.6 cm diameter. The dividers are perforated allowing sensory but no physical contact between the pair of mice.</td>
		</tr>
		<tr>
			<td>Clear rectangular rat cages&#160;</td>
			<td>Allentown</td>
			<td></td>
			<td>24 cm (w) &#215; 48.3 cm (d) &#215; 22.23 (h).</td>
		</tr>
		<tr>
			<td>Cotton squares for bedding</td>
			<td>Inotiv Envigo</td>
			<td>T.6060 iso-BLOX</td>
			<td>2.5 cm x 2.5 cm. Added to the social defeat apparatus.</td>
		</tr>
		<tr>
			<td>Hard woodchip bedding</td>
			<td>Inotiv Envigo</td>
			<td>Teklad 7090, 7115</td>
			<td>Sani-chip bedding.</td>
		</tr>
		<tr>
			<td>Large binder clips to secure the steel-wire tops</td>
			<td>STAPLES</td>
			<td>Item #: 132429, Model #: 24178-CA</td>
			<td>51 mm</td>
		</tr>
		<tr>
			<td>Medium binder clips to secure the steel-wire tops</td>
			<td></td>
			<td>Item #: 132367, Model #: 24172-CA</td>
			<td>32 mm, in case the cover lids of the rat cages do not close with the large clips</td>
		</tr>
		<tr>
			<td>Pain relief cream</td>
			<td>Polysporin</td>
			<td></td>
			<td>Plus Pain Relief Cream (red format, NOT ointment), 2 Antibiotics plus lidocaine hydrochloride</td>
		</tr>
		<tr>
			<td>Paired Steel-wire tops&#160;</td>
			<td></td>
			<td></td>
			<td>24 cm (w) &#215; 48 cm (d) with 0.6 cm (w) of separation between the grill</td>
		</tr>
		<tr>
			<td>Removable wire-mesh enclosure&#160;</td>
			<td>Johnston industrial plastics</td>
			<td></td>
			<td>11 cm (w) &#215; 6.8 cm (d) &#215; 42 cm (h) custom order; two per social interaction test arena secured in precut clear polycarbonate</td>
		</tr>
		<tr>
			<td>Social interaction open-field arena</td>
			<td>PEXIGLAS</td>
			<td></td>
			<td>45 cm (w) &#215; 45 cm (d) &#215; 49 cm (h), custom-crafted from opaque acrylic glass (Plexiglas)&#160;</td>
		</tr>
		<tr>
			<td>Stopwatch&#160;</td>
			<td></td>
			<td></td>
			<td>For timing defeat sessions</td>
		</tr>
		<tr>
			<td>Video camera with infrared lights&#160;</td>
			<td>Swann</td>
			<td>SRDVR-44580V&#160;</td>
			<td>Swann Camera - 4 Channel 1080p Digital Video Recorder &#38; 2 x PRO-T853</td>
		</tr>
		<tr>
			<td>Video tracking software&#160;</td>
			<td>Topscan</td>
			<td></td>
			<td>2.0 Clever Systems Inc.</td>
		</tr>
	</tbody>
</table>

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.

A total of four different experiments were performed using the chronic social defeat stress model in adolescent C57BL/6 male mice (PND 21). However, this model presented important limitations for its use in early adolescent C57BL/6 mice.
Equipment required modifications for adolescent C57BL/6 mice 
The first limitation was that the equipment used for the social defeat apparatus was designed for adult mice. Because of their size, adolescent C57BL/6 mice at PND 21 were able...

Watch this Scientific Journal Video about Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice at JoVE.com

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 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 ...

social-defeat-stress-model-for-adolescent-c57bl6-male-and-female-mice

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Neuroscience

1.2K Views.  McGill University. 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. 

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

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, at a subliminal perceptual level, and related brain mechanisms can be experimentally isolated by presenting the stimuli quickly. Subliminal presentation of faces that belong to different ethnicity groups, races, or gender has been shown to be successful in investigating social implicit responses. However, many implicit responses are based on knowledge previously gained about the faces (e.g., sexual orientation, political views, and socioeconomic status) and not solely on physical appearance. Here, a novel method called post-movie subliminal measurement (PMSM) is presented. When watching a socially engaging movie, a spectator gains knowledge about the protagonist and becomes familiar with his/her identity and world views. When the face of the protagonist is presented subliminally after the movie, it evokes an implicit neural response depending on what is learned about the protagonist. With a vast number of movies available, each depicting a variety of people with different identities, the PMSM method enables investigation of the brain&#39;s complex implicit biases in a manner that resembles real-life social perceptions.

Post-Movie Subliminal Measurement PMSM, for Investigating Implicit Social Bias

This protocol describes the use of movies to investigate brain mechanisms underlying implicit social biases during functional magnetic resonance imaging. When the face of a protagonist is presented after a movie subliminally, it evokes an implicit response based on knowledge of the protagonist gained during the movie.

New knowledge is continuously gained from a social environment that can influence how people respond to each other. Such responses often occur implicitly, ...

Social Defeat Stress Model for Adolescent C57BL/6 Male and Female Mice

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Low-stress Route Learning Using the Lashley III Maze in Mice

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Assessment of Social Interaction Behaviors

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias