This study was supported by the American Heart Association (IRG 14330015, PRE 16850005, PRE 20380386, EIA 0740129N), and the National Institutes of Health (RR 2865AR; P20 RR 016477).

All procedures described below have been reviewed and approved by the Institutional Animal Care and Use Committee at the West Virginia University Health Sciences Center.
1. Selection of Animal Model&#160;

NOTE: Most commonly used species in the UCMS model include Sprague Dawley, and Wistar rats and the BALB/cJ mouse; other murine models have shown limited efficacy (DBA/2, C57BL/6).&#160; However, careful consideration should be given to the particular mouse or rat strain of choice, as the genetic background and susceptibility to environmental stressors vary widely among species and have been shown to be critical factors affecting the outcomes of the study11,14,15,19.
<ol>
	<li>Use animals of the appropriate age (e.g.,&#160;if the desired endpoint is to be evaluated in a 17-week old rat, then the rats must start the UCMS protocol at ~9 weeks of age) as this protocol takes at least 7-9 weeks to fully induce depressive symptoms.</li>
	<li>Single-house animals undergoing UCMS in IACUC-approved cages and facilities, with food and standard chow available ad libitum (unless otherwise required by specific protocol).</li>
	<li>Take special care to ensure controls are housed in a quiet room with minimal activity to reduce effects of any possible unknown environmental stressors.</li>
</ol>
2. Unpredictable Chronic Mild Stress Protocol&#160;
NOTE: Animals undergoing UCMS are transported daily (&#62;5 days per week) to a clean room used for UCMS manipulations. Animals are exposed to 1 or 2 of the stressors listed below each day. Stressors are performed on a randomized schedule, and each stress is administered for a minimum of 3-4 hr (see Table 1).
<ol>
	<li>Dampen the bedding by pouring 10-20 oz. of clean water into each standard cage. Adjust the amount of water accordingly to the size of the cage in order to fully dampen the bedding but not to cause pooling of water. Reside the animals in damp cage for 3-4 hr.</li>
	<li>Remove bedding from each cage for 3-4 hr, after which animals are transitioned into another stressor requiring an empty cage (shallow bath, cage tilt) or placed into a clean cage with fresh bedding.</li>
	<li>Tilt cages to approximately 45&#176; (without bedding) for 3-4 hr. Cages can be tilted with a sturdy object that will remain in place as the animal moves around in the cage.</li>
	<li>Alter normal 12 hr/12 hr light/dark cycles into successions of 30 min periods lasting for 8 hr. After, return animals to the normal cycle.</li>
	<li>Transfer each animal from its home cage to that of the cage of a neighboring animal that has been removed for 3 hr.</li>
	<li>Remove all bedding from each cage and add water to a depth of ~0.25 inches for mice or ~0.5 inches for rats for approximately 4 hr. Use water that is warmer than RT (~30 &#176;C) to minimize hypothermia potential. Briefly dry animals with a soft towel at the conclusion of the water exposure prior to placement into clean cages.</li>
	<li>Expose animals to predator smells/sounds by adding randomly distributed sample tufts of fur or urine (10-20 ml) from a natural predator species into the cage for 3-4 hr. Alternatively, play growling or predatory noises from natural predator species in close proximity to cages for 3 hr.</li>
	<li>At the end of each daily stress period, place all animals into clean cages and return them to the housing facility.</li>
</ol>
3. Animal Monitoring/Grooming Scores
<ol>
	<li>Inspect each animal daily for the development of wounds or sores that may require veterinary treatment. Animals that develop mild injuries may continue in the UCMS protocol on the consent of the veterinarian.
	<ol>
		<li>Two investigators trained in evaluating coat grooming inspect each animal and assign a weekly coat score on a scale of 0-1. This score is determined by assigning individual scores (0- clean, 1-dirty) to each of 8 body regions- head, neck, dorsal coat, ventral coat, tail, forelimbs, hind limbs, and genital region. The same investigators are to perform this task throughout the UCMS protocol to maintain consistency in assigning coat scores.</li>
		<li>Any animal that experiences a sharp reduction in body weight (i.e., &#62;15%) should be reported to the veterinary staff and must be removed from the protocol.</li>
	</ol>
	</li>
</ol>
4. Behavioral Testing and Outcome Measurements&#160;
NOTE: Several methods of assessment can be used to quantify behavioral changes from the UCMS protocol in rodents.
<ol>
	<li>Cumulative Coat Grooming Score.
	<ol>
		<li>As described above, a weekly coat grooming score is assigned for each animal.</li>
	</ol>
	</li>
	<li>Sucrose Splash Test:
	<ol>
		<li>Place the animal in a clean cage lined with an cotton pad or towel to prevent bedding from adhering to its coat. Spray a 10% sucrose solution onto the head and dorsal coat of each animal and record total grooming behavior for 5 min.</li>
		<li>Grooming activity (defined as cleaning of the fur by licking or scratching) is measured by latency (idle time between the first spray and initiation of grooming) and frequency (number of times grooming a particular body part).</li>
	</ol>
	</li>
	<li>Sucrose Preference Test:
	<ol>
		<li>Habituate animals to drink 1% sucrose solution (w/v) 72 hr before the test and then provide exposure to two bottles (1% sucrose solution versus tap water in different bottles). After habituation, provide animals with ad libitum access to sucrose solution and tap water for 3 hr. After 3&#160;hr record the consumption volumes of sucrose solution and tap water and calculate sucrose preference as: [(sucrose consumption)/(water consumption + sucrose consumption)]&#215;100.</li>
	</ol>
	</li>
	<li>Tail Suspension Test (Mice Only):
	<ol>
		<li>Suspend mice by the tail from a stable rod (20 cm from the floor) using adhesive tape (2 cm from tip of tail). Record immobility time over 5 min. Mice are considered immobile only when they hang passively and completely motionless.</li>
	</ol>
	</li>
	<li>The Forced Swim Test:
	<ol>
		<li>Place animals in a container (~15-20 cm diameter for mice; ~55-60 cm diameter for rats) filled with water at a temperature of 26&#160;&#177;&#160;0.5&#160;&#176;C. Record the latency to the first bout of mobility and the time spent immobile during the 5-min testing period. At the end of a test, place the wet animal in a holding cage on a heating pad (30&#8211;35&#160;&#176;C) with normal bedding covered by an absorbent paper towel until dry.</li>
	</ol>
	</li>
	<li>Elevated Plus Maze:
	<ol>
		<li>Allow each animal to explore the maze freely for 5 min. Record total entries into each arm, and total time spent in each arm, according to a priori criteria20.</li>
	</ol>
	</li>
	<li>Locomotor Activity:
	<ol>
		<li>Record activity levels, defined as overall movement, fine movements, rearing movements, and inactivity, for both acute (30 min) and/or chronic (12 hr) time periods. Do this using the home cage or a novel environment, depending on the individual environment (although consistency should be maintained) in real time by an observer or post-hoc by replaying and analyzing camera footage.</li>
	</ol>
	</li>
</ol>
5. Expansion/Variation of UCMS Protocol
<ol>
	<li>Administer appropriate therapeutic agents or challenges to study their ability to either blunt the development of UCMS-induced outcomes (if initiated early) or to reverse severity of established UCMS-induced outcomes (if initiated late). This is dependent on the individual laboratory or protocol11.</li>
</ol>
6. Final Outcome Assessments
<ol>
	<li>Following the conclusion of UCMS protocols and behavioral testing, utilize animals in subsequent experiments in a manner that addresses the specific hypotheses, aims or goals of the individual laboratory. &#160; 
	NOTE: As examples, this can include cardiovascular, inflammatory or endocrine responses to chronic stress. Alternatively, therapeutic interventions can be introduced at this point to evaluate their effectiveness and mechanistic underpinnings.</li>
</ol>

<ol><li>NIMH Statistical Pages (Depression; Prevalence). , Available from:
 <a target="_blank" href="http://www.nimh.nih.gov/health/statistics/prevalence/major-depression-among-adults.shtml">http://www.nimh.nih.gov/health/statistics/prevalence/major-depression-among-adults.shtml</a> (2014).</li>
<li>NIMH Statistical Pages (Depression; Costs). , Available from:
 <a target="_blank" href="http://www.nimh.nih.gov/health/statistics/cost/index.shtml">http://www.nimh.nih.gov/health/statistics/cost/index.shtml</a> (2014).</li>
<li>Lett, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depression+as+a+risk+factor+for+coronary+artery+disease%3A+evidence%2C+mechanisms%2C+and+treatment%5BTitle%5D)+AND+%22Psychosom+Med%22%5BJournal%5D)">Depression as a risk factor for coronary artery disease: evidence, mechanisms, and treatment.</a> Psychosom Med. 66 (3), 305-315 (2004).</li>
<li>Plante, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depression+and+cardiovascular+disease%3A+a+reciprocal+relationship%5BTitle%5D)+AND+%22Metabolism%22%5BJournal%5D)">Depression and cardiovascular disease: a reciprocal relationship.</a> Metabolism. 54, 45-48 (2005).</li>
<li>Pizzi, C., Manzoli, L., Mancini, S., Bedetti, G., Fontana, F., Costa, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomic+nervous+system%2C+inflammation+and+preclinical+carotid+atherosclerosis+in+depressed+subjects+with+coronary+risk+factors%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">Autonomic nervous system, inflammation and preclinical carotid atherosclerosis in depressed subjects with coronary risk factors.</a> Atherosclerosis. 212 (1), 292-298 (2010).</li>
<li>Barden, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implication+of+the+hypothalamic-pituitary-adrenal+axis+in+the+physiopathology+of+depression%5BTitle%5D)+AND+%22J+Psychiatry+Neurosci%22%5BJournal%5D)">Implication of the hypothalamic-pituitary-adrenal axis in the physiopathology of depression.</a> J Psychiatry Neurosci. 29 (3), 185-193 (2004).</li>
<li>Bowman, R. E., Beck, K. D., Luine, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+stress+effects+on+memory%3A+sex+differences+in+performance+and+monoaminergic+activity%5BTitle%5D)+AND+%22Horm+Behav%22%5BJournal%5D)">Chronic stress effects on memory: sex differences in performance and monoaminergic activity.</a> Horm Behav. 43, 48-59 (2003).</li>
<li>Baune, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relationship+between+subtypes+of+depression+and+cardiovascular+disease%3A+a+systematic+review+of+biological+models%5BTitle%5D)+AND+%22Transl+Psychiatry%22%5BJournal%5D)">The relationship between subtypes of depression and cardiovascular disease: a systematic review of biological models.</a> Transl Psychiatry. 2, e92(2012).</li>
<li>O'Leary, O. F., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+translational+rodent+models+of+depression%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Towards translational rodent models of depression.</a> Cell Tissue Res. 354 (1), 141-153 (2013).</li>
<li>Mineur, Y. S., Belzung, C., Crusio, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+unpredictable+chronic+mild+stress+on+anxiety+and+depression-like+behavior+in+mice%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice.</a> Behav Brain Res. 175 (1), 43-50 (2006).</li>
<li>Yalcin, I., Belzung, I., Surget, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+strain+differences+in+the+unpredictable+chronic+mild+stress%3A+a+four-antidepressant+survey%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Mouse strain differences in the unpredictable chronic mild stress: a four-antidepressant survey.</a> Behav Brain Res. 193 (1), 140-143 (2008).</li>
<li>d'Audiffret, A. C., Frisbee, S. J., Stapleton, P. A., Goodwill, A. G., Isingrini, E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Depressive+behavior+and+vascular+dysfunction%3A+a+link+between+clinical+depression+and+vascular+disease%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Depressive behavior and vascular dysfunction: a link between clinical depression and vascular disease.</a> J Appl Physiol. 108 (5), 1041-1051 (2010).</li>
<li>Stanley, S. C., Brooks, S. D., Butcher, J. T., d'Audiffret, A. C., Frisbee, S. J., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effect+of+sex+on+chronic+stress-+and+depressive+behavior-induced+vascular+dysfunction+in+BALB%2FcJ+mice%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Protective effect of sex on chronic stress- and depressive behavior-induced vascular dysfunction in BALB/cJ mice.</a> J Appl Physiol. 117 (9), 959-970 (2014).</li>
<li>Ibarguen-Vargas, Y., Surget, A., Touma, C., Palme, R., Belzung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multifaceted+strain-specific+effects+in+a+mouse+model+of+depression+and+of+antidepressant+reversal%5BTitle%5D)+AND+%22Psychoneuroendocrinology%22%5BJournal%5D)">Multifaceted strain-specific effects in a mouse model of depression and of antidepressant reversal.</a> Psychoneuroendocrinology. 33 (10), 1357-1368 (2008).</li>
<li>Dalla, C., Pitychoutis, P. M., Kokras, N., Papadopoulou-Daifoti, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+animal+models+of+depression+and+antidepressant+response%5BTitle%5D)+AND+%22Basic+Clin+Pharmacol+Toxicol%22%5BJournal%5D)">Sex differences in animal models of depression and antidepressant response.</a> Basic Clin Pharmacol Toxicol. 106 (3), 226-233 (2010).</li>
<li>Mutlu, O., Gumuslu, E., Ulak, G., Celikyurt, I. K., Kokturk, S., Kır, H. M., Akar, F., Erden, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+fluoxetine%2C+tianeptine+and+olanzapine+on+unpredictable+chronic+mild+stress-induced+depression-like+behavior+in+mice%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">Effects of fluoxetine, tianeptine and olanzapine on unpredictable chronic mild stress-induced depression-like behavior in mice.</a> Life Sci. 91 (25-26), 1252-1262 (2012).</li>
<li>Gumuslu, E., Mutlu, O., Sunnetci, D., Ulak, G., Celikyurt, I. K., Cine, N., Akar, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+tianeptine%2C+olanzapine+and+fluoxetine+on+the+cognitive+behaviors+of+unpredictable+chronic+mild+stress-exposed+mice%5BTitle%5D)+AND+%22Drug+Res+%28Stuttg%29%22%5BJournal%5D)">The effects of tianeptine, olanzapine and fluoxetine on the cognitive behaviors of unpredictable chronic mild stress-exposed mice.</a> Drug Res (Stuttg). 63 (10), 532-539 (2013).</li>
<li>Isingrini, E., Belzung, C., Freslon, J. L., Machet, M. C., Camus, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluoxetine+effect+on+aortic+nitric+oxide-dependent+vasorelaxation+in+the+unpredictable+chronic+mild+stress+model+of+depression+in+mice%5BTitle%5D)+AND+%22Psychosom+Med%22%5BJournal%5D)">Fluoxetine effect on aortic nitric oxide-dependent vasorelaxation in the unpredictable chronic mild stress model of depression in mice.</a> Psychosom Med. 74 (1), 63-72 (2012).</li>
<li>Ripoll, N., David, D. J., Dailly, E., Hascoët, M., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antidepressant-like+effects+in+various+mice+strains+in+the+tail+suspension+test%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Antidepressant-like effects in various mice strains in the tail suspension test.</a> Behav Brain Res. 143, 193-200 (2003).</li>
<li>Komada, M., Takao, K., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elevated+plus+maze+for+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Elevated plus maze for mice.</a> J Vis Exp. (22), 1088(2008).</li>
<li>Golbidi, S., Frisbee, J. C., Laher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+stress+impacts+the+cardiovascular+system%3A+animal+models+and+clinical+outcomes%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">Chronic stress impacts the cardiovascular system: animal models and clinical outcomes.</a> Am. J. Physiol. Heart Circ. Physiol. , (2015).</li>
</ol>

The authors have nothing to disclose.

Extensive clinical and epidemiological evidence has indicated that chronic stress is one of the most potent precipitating factors for depression. Repeated exposure to stressors may potentiate individual vulnerability to depression and other neuropsychiatric disorders as a consequence of the psychological and physical demands within the body that accumulate over time. The use of validated preclinical animal models offers a valuable translational tool for studying depression and comorbid diseases. Firstly, an ideal animal model offers an opportunity to understand molecular, genetic and epigenetic factors that may lead to depression. By using such models, the underlying mechanisms and the causal relationship between genetic or environmental alterations and depression can be investigated, potentially allowing a more thorough insight into the pathophysiology of depression. Secondly, validated, improved models of depression are an indispensable tool for identifying and evaluating novel therapeutics and treatments, an essential first step towards developing improved diagnostic and treatment options. 
For many years, the UCMS model has been validated for use in psychological and behavioral studies as a translationally relevant rodent model for chronic stress, human depression and depressive symptoms, as it does an excellent job at reproducing clinical symptoms of human depression, including anhedonia and learned helplessness21. In addition, the temporal development of the depressive symptoms is also relevant for human disease conditions, as animals experiencing UCMS develop depressive symptoms over time in response to stress, which is considered to be more translationally relevant. The representative results reported here showcase the ability of this model to generate a symptomatic profile of depression, including both behavioral and physiological changes, in both a rat and mouse model. In addition, the severity of behavioral and physiological outcomes shows sex-specific differences that could potentially be used to study the pathological effects of stress on men and women21. &#160;
 It should be pointed out that the outcome of stress exposure is influenced by several variables under experimental conditions, including the nature of the stress (physical/systemic vs. cognitive/psychological), the severity of the stress, and exposure parameters. Different techniques can be used to measure depression-like behaviors and physiological outcomes and vary results among investigators21. For example, evaluation of the end points of behavioral tests may be variable depending on the use of recording methods as well as inconsistent methodology and bias amongst individual investigators. The particular strain/species used is also critical, as they have been shown to possess differences in susceptibility to UCMS. Further, other issues, such as the degree of control an animal has over stress exposure and the predictability of the stressor have been demonstrated to be important to the outcomes of the UCMS model, as greater unpredictability can reduce the probability of adaptive processes occurring upon repeated stress exposure and promote the appearance of stress effects on physiological and behavioral functions21.

Depressive mental illness is a complex neurological disorder that is currently recognized as a leading cause of disability and disease burden worldwide. The NIMH reports that approximately 12% of Americans suffer from clinical depression, with twice as many women affected versus men1. In the US alone, depression accounts for billions of dollars direct healthcare costs and an estimated $193 billion more in indirect costs (lowered earnings and lost productivity)2. Symptoms of depression include anhedonia, changes in weight and sleep cycles, decreased physical activity and personal hygiene, feelings of hopelessness or guilt, and/or reoccurring thoughts of death or suicide. During the last decade, epidemiologic and clinical studies have indicated that depression is an independent risk factor for cardiovascular disease (CVD) morbidity and mortality3, and is predictive of more severe prognosis of cardiovascular pathologies, including atherosclerosis, hypertension, myocardial infarction, and coronary artery disease, regardless of prior history of overt CVD4. Despite the increasing prevalence and adverse public health impact of depression, the etiology and related pathophysiology of this disease is poorly understood and the heterogeneity of the disorder due to various factors (e.g., genetic, biological, and environmental components) has made clinical diagnosis difficult to define.
Evidence indicates that irresolvable psychological stress is a major contributing factor for developing depressive illnesses and may also be a potent pathogenic factor linking depression and CVD, in part due to disruption and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis5,6. Dysfunction of the HPA axis is a major mechanism that has been linked to the behavioral and physiological changes observed in depression and the development of several CVD risk factors, including dyslipidemia, obesity, and diabetes7. Multiple preclinical models of depression have been developed in attempt to replicate the mechanism of altered HPA axis activity characteristic of clinical depression; such models provide a validated means for investigating the behavioral, neurological, and physiological changes associated with chronic and acute stress in animals. The validity of an animal model of disease is based on the relevance of the etiology and progression of the model design and its ability to recapitulate anatomical, neurophysiological, and behavioral features observed in human disease. In addition, preclinical responses to treatments (such as SSRIs) should yield similar results to those observed in clinical settings.
Several animal models of stress-induced depression are currently utilized in research, such as learned helplessness, early life stress, and social defeat stress. However, each of these models has inherent disadvantages that lessen their translational efficacy8. Within the last decades, the Unpredictable Chronic Mild Stress (UCMS) protocol has emerged as one of the most translationally-relevant models for studying the pathophysiology of depression in rodents9. This model is based on the fundamental concept that chronic exposure to stressors disrupts stress response systems and ultimately leads to the development of depressive disorders. During the UCMS protocol, animals are exposed to a randomized series of mild environmental and social stressors on a daily basis. A crucial factor that enhances the relevance of this model to human situations lies in the high degree of unpredictability and uncontrollability of the stressors, as well as the time at which they are introduced. Additionally, the UCMS protocol uses only mild stressors, instead of relying on early life or aggressive physical stimuli. Over a period of UCMS exposure, depressive behaviors develop and are comparable to clinical symptoms, including decreased responsiveness to rewards (anhedonia), changes in physical activity and investigative behavior (helplessness and despair), deterioration of the coat state and altered sexual activity10. Almost all demonstrable symptoms of depression have been reported using this model, and studies have demonstrated that these behaviors persist for several weeks following the cessation of stress. In addition, these UCMS-induced depressive behaviors can gradually be revered by chronic, but not acute, treatment with particular antidepressants, suggesting similar neurological effects of therapeutic improvement that closely mirrors the clinical action and variable efficacy of these agents in humans11,14-18 . Here, we report a detailed description of the UCMS protocol and describe typical behavioral and vascular outcomes in mice.

<table><tbody><tr><td>Temporary animal cages with lids</td><td></td><td></td><td>Provided by your animal care facility</td></tr><tr><td>Rodent bedding</td><td></td><td></td><td>Provided by your animal care facility</td></tr><tr><td>Predator fur or urine</td><td></td><td></td><td>Provided by your animal care facility</td></tr><tr><td>Rodent cage drinking bottles</td><td></td><td></td><td>Provided by your animal care facility</td></tr><tr><td>1,000 ml graduated cylinder</td><td>variable</td><td>variable</td><td>This is optional.&amp;nbsp; Any container from which a known quantiity of water can be poured will be appropriate</td></tr><tr><td>Wooden blocks cut from 2x4</td><td>variable</td><td>variable</td><td>8 inch sections are cut to facilitate cage tilt procedures</td></tr><tr><td>Soft paper towels</td><td></td><td></td><td>Provided by your institution</td></tr><tr><td>Small spray bottle&amp;nbsp;</td><td>Walmart</td><td></td><td>100-200 ml volume is sufficient, used for sucrose splash test</td></tr><tr><td>Medium (mice) or large (rats) plastic tubs for swim testing</td><td>Walmart</td><td>variable</td><td>Should be of sufficient depth that the animals cannot touch bottom (&lt;I&gt;e.g.&lt;/I&gt;, 2 feet)</td></tr></tbody></table>

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.

An example of the schedule for one week following the UCMS procedure is shown in Table 1. Each week, stressors were randomized to prevent acclamation and to ensure unpredictability of the stress challenge each day.
Measures of Depressive like Behaviors
Following 8 weeks of UCMS, there were significant alterations to both behavioral and physiological outcomes in chronically stressed rodents relative to their non-stressed control strains. Development of depressive like symptoms following UCMS was assessment via coat score, sucrose splash test, and tail suspension (mice only; references12,13). A progressive decline in coat grooming scores was observed in all UCMS animals (Figure 1, Panel A), and this marked degradation in the physical state of the coat suggests decreased grooming activity. This behavior may parallel the lack of motivation to or loss of interest in performing everyday tasks, such as the maintenance of minimal personal care.
UCMS animals also demonstrated an increased latency to groom and reduced total grooming time in the sucrose splash test (Figure 1, Panels B and C). Increased time to initiate grooming behavior along with decreased time spent grooming is associated with reduced motivation and a decline in self stimulatory behavior, or reduced sensitivity to pleasure. This is representative of the core symptom of depression, anhedonia. In addition, there was a significant increase in the immobility time during the tail suspension test in mice. As stated previously, this behavior is a measure of behavioral despair with greater immobility representing a more severe depressive like symptom.
Measures of Physiological Changes
MAP can be significantly elevated as a result of the UCMS protocol, but this is not consistently observed (Table 1). A significant reduction in the dilator response to methacholine was observed in the aortic rings of UCMS BALB/cJ mice compared to controls (Figure 2). This increased shift towards blunted dilator responses is observed in clinical cases of increased risk for development of cardiovascular disease.
UCMS mice manifested increased plasma levels of cortisol, a marker of chronic physiological stress, and nitrotyrosine, a marker of chronic oxidative stress (Table 2).
Additionally, UCMS mice showed significantly elevated levels of the pro-inflammatory markers TNF-&#945; and MCP-1. Together, these results suggest that stressed animals develop a pathophysiological disease profile following 8 weeks of UCMS (Table 2).
Divergence with Sex
Recent evidence has suggested that there is a substantial quantitative divergence in the responses to UCMS between male and female mice (13). Specifically, data suggest that female mice, exposed to the identical UCMS protocol as age-matched males, develop more severe behavioral responses to the protocol than do males (Table 3 and Figure 3), with elevated levels of cortisol, nitrotyrosine and markers of inflammation.
However, despite this divergence between sexes following the UCMS protocol, the vasculopathy that develops as a result appears to be blunted in the female sex as compared to the males, strongly suggesting a superior maintenance of endothelial function, despite the compromised environment (Figure 4).
For Figures 1 and 3, results were obtained using the techniques described above, with the application of ANOVA and t-tests as appropriate. For Figures 2 and 4, results were obtained using concentration-response relationships in ex vivo aortic ring preparations. Curves were fit with a three parameter logistic equation, with ANOVA and t-tests as appropriate for the lower bounds of the curves. Please see references12,13 for details.
<img alt="Figure 1" src="/files/ftp_upload/53109/53109fig1.jpg" /> 
Figure 1.&#160;Depressive Symptoms Following Eight Weeks of UCMS in Mice. Data are presented for coat status (Panel A), the latency (Panel B) and frequency (Panel C) of facial grooming following a 10% sucrose solution spray, and the total period of immobility during the tail suspension test (Panel D) for control and UCMS mice. * p &#60;0.05 versus control.Please see reference12 for details. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53109/53109fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53109/53109fig2.jpg" /> 
Figure 2. Vasodilator Responses to Methacholine from Mice Following Eight Weeks of UCMS. Data are presented for mice under control conditions (control lower bound = 10.3 &#177; 2.4%) and following eight weeks of the imposed UCMS protocol (UCMS lower bound = 33.6 &#177; 4.4%). * p &#60;0.05 versus responses in untreated vascular rings from control mice. Re-printed from reference12 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53109/53109fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53109/53109fig3.jpg" /> 
Figure 3. Differences in Depressive Symptoms Following Eight Weeks of UCMS in Male and Female Mice. Data are presented for coat status (A), the latency (B) and frequency (C) of facial grooming following a 10% sucrose solution spray, and the total period of immobility during the tail suspension test (D) for control and UCMS mice. * p &#60;0.05 versus Control in the sex; &#8224; p &#60;0.05 versus UCMS-Male. Re-printed from reference13 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53109/53109fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53109/53109fig4.jpg" /> 
Figure 4. Differences inVasodilator Responses from Male and Female Mice Following Eight Weeks of UCMS. Dilator responses of aortic rings to increasing concentrations of methacholine (upper panel) and sodium nitroprusside (lower panel) from mice under control conditions and after eight weeks of UCMS. * p &#60;0.05 versus Control in the sex; &#8224; p &#60;0.05 versus UCMS-Male. Re-printed from reference13 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53109/53109fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Sunday</td>
			<td>Monday</td>
			<td>Tuesday</td>
			<td>Wednesday</td>
			<td>Thursday</td>
			<td>Friday</td>
			<td>Saturday</td>
		</tr>
		<tr>
			<td>Stressor 1</td>
			<td>Alter Light/Dark</td>
			<td>Social Stress</td>
			<td>No bedding</td>
			<td>Damp Bedding</td>
			<td>Alter light/dark</td>
			<td>Predator sounds</td>
			<td>Social Stress</td>
		</tr>
		<tr>
			<td>Stressor 2</td>
			<td>Alter Light/Dark</td>
			<td>Cage Tilt</td>
			<td>Shallow Bath</td>
			<td>Cage Tilt</td>
			<td>Social Stress</td>
			<td>Shallow Bath</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Two sample weeks for imposition of unpredictable chronic mild stress (UCMS) protocol.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Control</td>
			<td>UCMS</td>
		</tr>
		<tr>
			<td>Mass (g)</td>
			<td>31 &#177; 2</td>
			<td>29 &#177; 4</td>
		</tr>
		<tr>
			<td>MAP (mmHg)</td>
			<td>84 &#177; 5</td>
			<td>93 &#177; 4</td>
		</tr>
		<tr>
			<td>Insulinplasma (ng/ml)</td>
			<td>1.3 &#177; 0.2</td>
			<td>3.9 &#177; 0.6*</td>
		</tr>
		<tr>
			<td>Glucoseblood (mg/dl)</td>
			<td>102 &#177; 8</td>
			<td>104 &#177; 7</td>
		</tr>
		<tr>
			<td>Cholesterolplasma (mg/dl)</td>
			<td>75 &#177; 8</td>
			<td>81 &#177; 8</td>
		</tr>
		<tr>
			<td>Triglyceridesplasma (mg/dl)</td>
			<td>102 &#177; 8</td>
			<td>128 &#177; 10*</td>
		</tr>
		<tr>
			<td>Nitrotyrosineplasma (ng/ml)</td>
			<td>17 &#177; 2</td>
			<td>27 &#177; 4*</td>
		</tr>
	</tbody>
</table>
Table 2. Baseline characteristics between mouse groups under control conditions and following 8 weeks of UCMS. All mice are aged 17-18 weeks. * p &#60;0.05 versus control. Modified from reference12.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Males</td>
			<td>Females</td>
			<td>UCMS-Males</td>
			<td>UCMS-Females</td>
		</tr>
		<tr>
			<td>Mass (g)</td>
			<td>29 &#177; 2</td>
			<td>28 &#177; 3</td>
			<td>30 &#177; 3</td>
			<td>29 &#177; 4</td>
		</tr>
		<tr>
			<td>MAP (mmHg)</td>
			<td>87 &#177; 4</td>
			<td>91 &#177; 4</td>
			<td>94 &#177; 5</td>
			<td>90 &#177; 5</td>
		</tr>
		<tr>
			<td>Insulinplasma (ng/ml)</td>
			<td>1.1 &#177; 0.3</td>
			<td>1.2 &#177; 0.4</td>
			<td>4.1 &#177; 0.7*</td>
			<td>4.5 &#177; 0.5*</td>
		</tr>
		<tr>
			<td>Glucoseblood (mg/dl)</td>
			<td>82 &#177; 7</td>
			<td>80 &#177; 8</td>
			<td>94 &#177; 8</td>
			<td>101 &#177; 10</td>
		</tr>
		<tr>
			<td>Cholesterolplasma (mg/dl)</td>
			<td>71 &#177; 7</td>
			<td>68 &#177; 8</td>
			<td>78 &#177; 6</td>
			<td>77 &#177; 10</td>
		</tr>
		<tr>
			<td>Triglyceridesplasma (mg/dl)</td>
			<td>94 &#177; 6</td>
			<td>101 &#177; 8</td>
			<td>109 &#177; 8</td>
			<td>116 &#177; 12</td>
		</tr>
		<tr>
			<td>Nitrotyrosineplasma (ng/ml)</td>
			<td>12 &#177; 3</td>
			<td>11 &#177; 4</td>
			<td>29 &#177; 5*</td>
			<td>36 &#177; 6*</td>
		</tr>
		<tr>
			<td>Cortisolplasma (pg/ml)</td>
			<td>12 &#177; 3</td>
			<td>14 &#177; 4</td>
			<td>29 &#177; 5*</td>
			<td>44 &#177; 4*</td>
		</tr>
		<tr>
			<td>TNF-aplasma (pg/ml)</td>
			<td>2.3 &#177; 0.3</td>
			<td>2.0 &#177; 0.2</td>
			<td>4.1 &#177; 0.4*</td>
			<td>6.4 &#177; 0.4*&#8224;</td>
		</tr>
		<tr>
			<td>MCP-1plasma (pg/ml)</td>
			<td>2.8 &#177; 0.3</td>
			<td>3.3 &#177; 0.5</td>
			<td>10.2 &#177; 1.0*</td>
			<td>14.8 &#177; 1.3*&#8224;</td>
		</tr>
	</tbody>
</table>
Table 3. Baseline characteristics between male and female mouse groups under control conditions and following 8 weeks of UCMS. All mice are aged 17-18 weeks. * p &#60;0.05 versus control. Modified from reference13.

Watch this Scientific Journal Video about An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents at JoVE.com

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

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 ...

an-unpredictable-chronic-mild-stress-protocol-for-instigating

Nanyang Technological University

Research

JoVE Journal

Behavior

22.4K Views.  West Virginia University Health Sciences Center. 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.

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

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

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

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

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

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of depression is essential. Because there are several types of depression, various animal models are needed for a deeper understanding of the disorder. Previously, a mouse model of subchronic and mild social defeat stress (sCSDS) using a modified chronic social defeat stress (CSDS) paradigm was established. In the paradigm, to reduce physical injuries from aggressors, the duration of physical contact between the aggressor and a subordinate was reduced compared to in the original CSDS paradigm. sCSDS mice showed increased body weight gain, food intake, and water intake during the stress period, and their social behaviors were suppressed after the stress period. In terms of the face validity of the stress-induced overeating and overdrinking following the increased body weight gain, the sCSDS mice may show some features related to atypical depression in humans. Thus, a mouse model of sCSDS may be useful for studying the pathogenic mechanisms underlying depression. This protocol will help establish the sCSDS mouse model, especially for studying the mechanisms underlying stress-induced weight gain and polydipsia- and hyperphagia-like symptoms.

Here, methods for developing a mouse model of subchronic and mild social defeat stress are described and used to investigate the pathogenic features of depression including hyperphagia- and polydipsia-like symptoms following increased body weight.

Stressful life events often increase the incidence of depression in humans. To study the mechanisms of depression, the development of animal models of ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Biochemistry

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 ...

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, ...

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

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

Neuroscience

Inducing Depression-like Behavior in a Mouse via Immobilization Stress

Source: Son, H., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59546">A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the method of inducing chronic stress in a mouse through repeated immobilization, leading to corticosterone release and reduced glutamate receptor activity in neurons, which results in depression-like behavior.