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>

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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.

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			<td>single cage</td>
			<td>Charles River Laboratories Japan</td>
			<td></td>
			<td>width [W] &#215; depth [D] &#215; height [H] = 143 &#215; 293 &#215; 148 mm</td>
		</tr>
		<tr>
			<td>M cage</td>
			<td>Natsume Seisakusho</td>
			<td></td>
			<td>W &#215; D &#215; H = 220 &#215; 320 &#215; 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&#8217;Hara &#38; 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

19.4K 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

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

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

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

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

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

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

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

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

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

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

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

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

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 ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

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 ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

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

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

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

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

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.