This work was funded by internal funds of the University Clinic Freiburg, Department of Psychiatry and Psychotherapy and the Berta-Ottenstein Program for Clinician Scientists (to SV). TS is funded by the grants of Medical Research Foundation (FRM) (AJE201912009450) and the University of Strasbourg Institute of Advance Studies (USIAS) (2020-035), as well as Centre National de la Recherche Scientifique (CNRS), France.

All experiments were performed in agreement with European guidelines (EU 2010/63) and in accordance with the German animal protection law (TierSchG), FELASA (www.felasa.eu/guidelines.php), the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html) guide for the care and use of laboratory animals, and were approved by the animal welfare committee of the University of Freiburg and by the Comite d&#8217;Ethique en Matiere d&#8217;Experimentation Animale de Strasbourg (CREMEAS, CEEA35), as well as local authorities. Both sexes of C57Bl6N wild-type mice aged 10-14 weeks (70-98 post-natal days, PND) were used for wild-type (WT) indicated experiments. As a stress-resilient line, the transgenic mouse line with enhanced expression of adenosine A1 receptors under the forebrain neuronal CaMKII promoter was used9,15. After the experiments, mice were sacrificed by cervical dislocation.
1. Preparation
<ol>
	<li>Obtain an animal research license, including thorough experimental planning.</li>
	<li>Arrival: On arrival, raise the animals in the animal facility to perform the CDM. If the animals are bought from an external supplier, allow them at least 2 weeks to adapt to the new environment.</li>
	<li>Housing: To house the animals, ensure that the cages are not occupied with the maximum number of animals to avoid additional stress. Guarantee that housing conditions are in line with international recommendations of mouse housing (for further information, see22) and constantly maintain them at all times. 
	NOTE: The most important standard housing conditions include individually ventilated cages with 25-120 air changes per hour, 12 h light-dark cycle, temperature as stable as possible (at least constant between 20-24 &#176;C), humidity as stable as possible (at least between 45%-65%), gnawing material and shelter present, no individual housing.</li>
	<li>Time point: Perform all experiments at the same time of the day. 
	NOTE: No direct assessment has been made to verify the influence of the daytime on CDM, but most behavioral tests evaluating depressive-like states show variations depending on the time of the day23,24,25, and it is highly probable that daytime also influences CDM.</li>
	<li>Nesting material: Reduce the nesting material to a minimum. Ensure there are no running wheels, etc., present in the cage. 
	NOTE: Enriched environment prevents the induction of a depressed state.</li>
	<li>Group composition: Allow the animals to remain in the same group throughout the whole experiment. Group the female mice together even from different litters; group the male mice together with littermate male animals. Due to upcoming aggressiveness, especially of males, biting and barbering may become a problem, therefore give special emphasis to the group composition. Avoid single housing as deprivation is a major additional stressor.</li>
	<li>Animals: Use different mouse strains, even though specific differences have been observed9,10. A frequently used mouse strain is C57Bl6N. Label mice in order to perform paired statistical analysis (see step 3.2.4).</li>
	<li>Animal sex: Equally use both male and female mice.</li>
	<li>Animal age: Ensure that the animals are at least 10 weeks (70 PND) old. Do not use younger animals due to the exhaustion caused by swimming.</li>
	<li>Equipment: Use a transparent glass cylinder/beaker with a capacity of at least 2 L, a diameter of 24-26 cm, and a minimum height of 30 cm. Further requirements include a thermometer to check the water temperature, paper towels, red light heating lamp/heating mat or comparable source of heating, timer, stopwatch, quiet surroundings. Videotape the swim sessions for offline analysis and documentation. Ensure that the date and the time are continuously visible on the tape/file, together with an identification code number for the individual animal. Store the files for later analysis and further reference. Film from the side of the glass cylinder, not from above, to facilitate analysis.</li>
</ol>
2. Induction phase
<ol>
	<li>Before starting
	<ol>
		<li>Visually observe the animals for abnormalities, including signs of biting or barbering. Exclude the whole cage from the experimental series if an animal shows any minimal injuries. Ensure that a veterinarian is available at any time as injuries will worsen during the experiment and will prevent continuation as mice become more aggressive under the influence of stress.</li>
		<li>Obtain the bodyweight for each animal before starting the experiment. Ensure that the weight loss often observed does not exceed 20% of the initial body weight. Exclude animals with a weight loss of over 20% and immediately euthanize them due to the assumed high suffering.</li>
		<li>Fill up a beaker or cylinder with water at room temperature (22-23 &#176;C) to a height of at least 20 cm from the bottom, leaving a minimum of 10 cm between the water surface and the upper border of the vessel.</li>
	</ol>
	</li>
	<li>Performance
	<ol>
		<li>Gently transfer the animals into the water. During the swim phase, keep the animal under continuous observation to prevent drowning. Observe from a position where the animal cannot see the experimenter (for instance, video observation from a room next door).</li>
		<li>Set a chronometer at the beginning of the experiment. Take the animals out of the water after 10 min by simply grabbing their tails. Gently dry them with a paper towel and put them either under a heating light or on a heating mat.</li>
		<li>Evaluate only one animal at a time. Ensure that animals cannot see each other (for example, separate the housing cage from the experimental set-up by a room-divider).</li>
		<li>Perform the forced swim session for 10 min each day for 5 consecutive days.</li>
	</ol>
	</li>
	<li>Finishing
	<ol>
		<li>Transfer the animals back to their home cages after five swim sessions and allow them to rest for at least 2 days. Start specific treatment interventions subsequently.</li>
	</ol>
	</li>
</ol>
3. Evaluation of an anti-depressive treatment
<ol>
	<li>Time course
	<ol>
		<li>Assess the acute and subchronic treatments with the CDM. Depending on the scientific question, adapt the resting period between the induction phase and the read-out.</li>
		<li>To evaluate the acute and rapid-acting potency of ketamine, choose a short resting period (a few days) after the induction phase of CDM. Apply the treatment (i.e., intraperitoneal injection), and then perform the evaluation (additional swim session or different evaluation method) shortly afterward.</li>
		<li>To evaluate the effects of a subchronic treatment, increase the treatment period up to 4 weeks (there is no data available for longer treatment periods). For example, give the oral treatment with imipramine to the animals during 4 weeks after the induction phase and evaluate thereafter.</li>
		<li>Start to evaluate the depressed state right after the end of the treatment period, e.g., the following day. Always choose an identical time period for control and experimental conditions.</li>
	</ol>
	</li>
	<li>Immobility time
	<ol>
		<li>Proof-of-concept
		<ol>
			<li>To use immobility time as a read-out method, evaluate each day of the induction phase and the test day to provide a proof-of-concept (see Figure 1). For further experimental series, reduce the assessments to day 1, day 5, and the test day (see Figure 1C).</li>
			<li>Videotape each experiment. Allow two trained observers who are blinded to the experimental conditions to perform the analysis independently. Video analysis enables the experimenter to observe the behavior from a different room, therefore minimizing the interference with the test (for example, see the video file in the supplementary material).</li>
		</ol>
		</li>
		<li>Conditions: Observe and identify the three different behavioral conditions during the swim test: struggling, swimming, and immobility. Most researchers focus on immobility; a further differentiation between struggling and swimming is rarely useful and dramatically increases the complexity and duration of the analysis.
		<ol>
			<li>Struggling: The animal actively tries to escape from the threatening situation. This involves pawing the side of the cylinder with the head oriented toward the wall and movements of all limbs. The water surface is typically slightly turbulent.</li>
			<li>Swimming: The animal moves at least both hind paws and travels a distance throughout the water. It actively searches for a way out but does not try to overcome the glass wall of the vessel. Swimming does not involve lifting the paws above the water surface, and the body is usually oriented parallel to the walls of the cylinder. In this condition, animals frequently turn around or move in circles.</li>
			<li>Immobility: The animal keeps still, in a freezing-like position, and does not move at all or only moves the tail, or the forepaws to keep its head above the water surface. No distance is actively traveled except for passive floating, and no directed movement of the front paws is observed.</li>
		</ol>
		</li>
		<li>Tracking
		<ol>
			<li>Perform the assessment using offline video recordings. Use blinded ratings by two independent and experienced examiners and calculate averages between the two ratings.</li>
			<li>Repeat the ratings if the results of the two raters differ above a previously determined range. Continuously observe the mice as the different conditions frequently change between struggling, swimming, and immobility.</li>
			<li>Use a stopwatch to measure the total time spent in a focused stage (usually immobility) over the 10 min the mouse stays in the water. Consider a short latency of about a second before changing the ongoing time measurement (e.g., if an animal remains for 20 s in immobility and only moves once for less than a second and returns to immobility for another 10 s, the total immobility time is 30 s).</li>
		</ol>
		</li>
		<li>Statistics: Due to the relatively high inter-individual standard deviation (probably caused by a transfer of hierarchy-depending behavior from the cage to the swim test), mark or label the animals to perform paired (instead of unpaired) parametric tests afterward. Evaluate the normality distribution and, depending on the specific question, perform analysis of variance (ANOVA) with post-hoc t-tests or paired t-tests to compare the different groups. Perform the analysis using absolute values of immobility time (s) or as normalized values.
		<ol>
			<li>Absolute values: Give mean values of the immobility time from day 1 to day 5 and for the test day &#177; SEM (see Figure 1A). Compare the averaged values for day 1 and day 5, preferably using a paired t-test to validate the induction of a depressed state. If there is a significant difference between day 1 and 5, compare the mean values of day 5 to the averaged results of the test day. Ensure that a typical group size in one experiment is between 6 and 10 animals and expect significant differences between baseline and post-induction immobility times in wild-type animals. Comparing different groups with an unpaired t-test is difficult if absolute values are used because of baseline differences; therefore, use normalized values.</li>
			<li>Relative/Normalized values: Compare the different treatment effects by normalization to the individual result on day 5, and then express the values as a percentage of day 5 (see Figure 1B).</li>
		</ol>
		</li>
		<li>Control experiments 
		NOTE: The swimming performance might be correlated with locomotion. Substances that cause a hyper-locomotion could induce false-positive results (namely, a decrease of immobility time); as well as sedative agents could artificially increase immobility time.
		<ol>
			<li>Evaluate the changes in locomotion for unknown substances before performing the swim analysis. Use Open Field Test (OFT) in a separate group of animals for at least 10 min.</li>
			<li>Choose the same observation time (10 min) in the OFT as in the CDM to detect unspecific hyper-locomotive effects of the tested compound that might influence CDM read-out via measurement of immobility-time with high validity.</li>
			<li>In case of significant hyper-locomotive effects, do not evaluate the swim session to assess the anti-depressive potency but use different read-out methods (for instance, sucrose preference, tail suspension test, etc.).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Evaluation of the development of a depressive-like state
<ol>
	<li>To evaluate the development of a depressive disorder, assess each day of the induction phase to measure the immobility-time. 
	NOTE: In this case, a minor increase of immobility-time between each day describes resilience, whereas a stronger and earlier increase compared to untreated or wild-type animals represents an enhanced vulnerability to stress-induced despair. By treating mice before the swimming event, the preventive intervention or transgenic mouse lines could be assessed concerning the development of behavioral despair.</li>
</ol>

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All the authors declare no conflicts of interest.

The CDM model represents a relevant and established model for testing the anti-depressive potency of new interventions and opens an extended time window for molecular or electrophysiological experiments to elucidate the pathophysiology of depression. Especially when combined with other tests to assess a depression-like state, CDM has a high face and concept validity. It combines subchronic stress and acquired helplessness for induction and produces a long-lasting depressive-like state. It is insensitive to the single app...

Affective disorders, such as major depressive disorder, are among the most frequent and challenging mental illnesses and are associated with high individual suffering1, an increase of suicide risk2, and cause a considerable socioeconomic burden3 for society. Despite its impact, treatment options are limited, and there is an urgent need for the development of novel antidepressive interventions, especially due to the innovation crisis in psychopharmacology over the last decades. In order to understand the pathophysiology of depression and test potential new agents, rational and valid animal models are urgently needed4. For almost half a century, the classical forced swim test (FST), originally described by Porsolt5, was used as induction and read-out for screening of potential novel antidepressants. It consists of a forced swim period for 5-15 min on day 1, subsequent one-time drug application, and evaluation of the portion mice spend immobile in water in another swim period on the following day. The immobility time was considered to represent a missing natural escape behavior and was thought to correlate with the degree of a depression-like state in the mice5.
The classical FST has been heavily criticized, not only in the scientific community6,7,8 but also in public media8. Most controversies around the FST are due to the short induction and treatment periods of only 1 day in the classical paradigm. It was argued that FST represents rather an acute trauma model than a state comparable to human depression. Moreover, the Porsolt test might be suitable as a screening tool for potential antidepressive agents, but it ignores the delayed onset of action of many antidepressants.
The chronic despair model (CDM)9,10,11,12,13,14,15, which is derived from the original FST, represents a more appropriate animal model for depression. In CDM, repeated swim stress over 5 consecutive days avoids acute traumatic effects. By failing to escape from a repeated and ongoing stressful situation, mice are thought to develop a state of helplessness, surrender, and ultimately despair. This paradigm is more comparable to current psychological theories for the development of depression in humans than a single acute trauma, which is commonly experienced at the onset of a posttraumatic stress disorder. The resulting depression-like state in CDM is stable for up to 4 weeks9 and therefore opens the possibility for longer treatment periods, which are better comparable to clinical conditions, where antidepressants usually need 2-4 weeks to show a benefit16.
The evaluation of the depressive-like state should then be multidimensional. The measurement of immobility time, such as in the classical FST, is useful, but should not be used as the only outcome parameter. Various methods, which are described below, should be able to map different dimensions of a depressive state in line with symptoms usually found in depressed humans. Suitable read-out assessments could include escape behavior (immobility time9,10,17), tail suspension test (TST)9, anhedonia (classical sucrose preference test (SPT)18), motivation-oriented behavior (nose-poke sucrose preference test (NPSPT)10), expectation/exploration-behavior (response to ambiguous signal19; Y-maze test9), electrophysiology (measurements of long-term plasticity (long-term potentiation, LTP; long-term depression, LTD)20), molecular assessments (activation patterns of immediate early genes (IEGs); further stress patterns21).
Theoretically, a repeated swim test can be used to induce a depressed state without any assessment of immobility time. However, it is strongly recommended to provide at least a proof-of-concept experimental series with immobility times. Additionally, CDM represents a suitable model to assess the development of a depressive-like state by measuring immobility time during the induction phase. Specific mouse strains or mice treated before swimming can be evaluated with respect to resilience or vulnerability to stress and the induction of behavioral despair.

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animal models of depression - chronic despair model (cdm)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

In the first swim session of the induction phase of CDM, mice usually show a mean immobility time between 190 s and 230 s, which constantly rises with every additional swim session (Figure 1A). This increase is more pronounced in the first 3 days and reaches a plateau-like phase during the last 2-3 days. The immobility-time measured on day 5 remains stable over up to 4 weeks, indicating stable behavioral despair. The antidepressant potency of an intervention ...

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Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...

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6.9K Views.  University of Freiburg. The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Video: Animal Models of Depression - Chronic Despair Model CDM

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 ...

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

A New Method for Inducing a Depression-Like Behavior in Rats

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is no existing format for studying the mechanism of action, prevention, containment, and treatment of contagious depression. The purpose of this study, therefore, was to establish the first animal model of contagious depression.
Healthy rats can contract depressive behaviors if exposed to depressed rats. Depression is induced in rats by subjecting them to several manipulations of chronic unpredictable stress (CUS) over 5 weeks, as described in the protocol. A successful sucrose preference test confirmed the development of depression in the rats. The CUS-exposed rats were then caged with na&#239;ve rats from the contagion group (1 na&#239;ve rat/2 depressed rats in a cage) for an additional 5 weeks. 30 social groups were created from the combination of CUS-exposed rats and na&#239;ve rats.
This proposed depression-contagion protocol in animals consists mainly of cohabiting CUS-exposed and healthy rats for 5 weeks. To ensure that this method works, a series of tests are carried out - first, the sucrose preference test upon inducing depression to rats, then, the sucrose preference test, alongside the open field and forced-swim tests at the end of the cohabitation period. Throughout the experiment, rats are given tags and are always returned to their cages after each test.
A few limitations to this method are the weak differences recorded between the experimental and control groups in the sucrose preference test and the irreversible traumatic outcome of the forced swim test. These may be worth considering for suitability before any future application of the protocol. Nonetheless, following the experiment, na&#239;ve rats developed contagion depression after 5 weeks of sharing the same cage with the CUS-exposed rats.

This protocol describes a new model by which healthy rats could contract depression over a given time periodthrough contagion by exposure to chronic unpredictable stressed (CUS) rats.

Contagious depression is a phenomenon that is yet to be fully recognized and this stems from insufficient material on the subject. At the moment, there is ...

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

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 Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

Generating Acute and Chronic Experimental Models of Motor Tic Expression in Rats

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic generation is associated with abnormal inhibition of the basal ganglia, particularly its primary input structure, the striatum. In animal models of both rodents and non-human primates, local application of GABAA antagonists, such as bicuculline and picrotoxin, into the motor parts of the striatum induces local disinhibition resulting in the expression of motor tics.
Here, we present acute and chronic models of motor tics in rats. In the acute model, bicuculline microinjections through a cannula implanted in the dorsal striatum elicit the expression of tics lasting for short time periods of up to an hour. The chronic model is an alternative enabling the extension of tic expression to periods of several days or even weeks, utilizing continuous infusion of bicuculline via a sub-cutaneous mini-osmotic pump.
The models enable the study of the behavioral and neural mechanisms of tic generation throughout the cortico-basal ganglia pathway. The models support the implantation of additional recording and stimulation devices in addition to the injection cannulas, thus allowing for a wide variety of usages such as electrical and optical stimulation and electrophysiological recordings. Each method has different advantages and shortcomings: the acute model enables the comparison of the kinematic properties of movement and the corresponding electrophysiological changes before, during and after tic expression and the effects of short-term modulators on tic expression. This acute model is simple to establish; however, it is limited to a short period of time. The chronic model, while more complex, makes feasible the study of tic dynamics and behavioral effects on tic expression over prolonged periods. Thus, the type of empirical query drives the choice between these two complementary models of tic expression.

We present protocols for generating acute and chronic experimental models of tic expression in freely behaving rats. The models are based on striatal cannula implantation and subsequent GABAA antagonist application. The acute model uses transient injections whereas the chronic model utilizes prolonged infusions via a subcutaneous implanted mini-osmotic pump.

Motor tics are sudden, rapid, recurrent movements that are the key symptoms of Tourette syndrome and other tic disorders. The pathophysiology of tic ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...