Name: a chronic immobilization stress protocol for inducing depression-like behavior in mice
Uploaded: 2019-05-15T14:45:00
Description: Watch this Scientific Journal Video about A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice at JoVE.com

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1A5A2008833 and NRF-2016R1D1A3B03934279) and the grant of lnstitute of Health Sciences (IHS GNU-2016-02) at Gyeongsang National University.

All experimental protocols and animal care were conducted according to the guidelines of the University Animal Care Committee for Animal Research of Gyeongsang National University (GLA-100917-M0093).
1. Materials
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	<li>Mice
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		<li>Use males of C57BL/6 strain background weighing 22&#8211;24 g at postnatal week 7. Habituate in the breeding room for 1 week before the experiments. 
		NOTE: All mice were purchased from a laboratory animal company.</li>
		<li>House mice individually ...

The complexity of the brain and heterogeneity of MDD make it challenging to create animal models that completely reproduce the condition. Many researchers have overcome this difficulty using an endophenotype-based approach32, in which anhedonia (lack of interest in rewarding stimuli) and despair are considered evolutionarily conserved and quantifiable behaviors in animal models, which are also seen in patients with depression33. In the present paper, we have presented a met...

Major depressive disorder (MDD) is the leading cause of mental disability worldwide, with an incidence that is increasing faster than anticipated. In 2001, the World Health Organization predicted that MDD would be the second most common disease in the world by 2020. However, it was already the second most common in 20131. In addition, current antidepressants have many limitations, including delayed effectivity, drug resistance, relapse, and various side effects2,3. Researchers must therefore develop more effective antidepressants. However, the ambiguous pathophysiology of MDD presents an obstacle to the development of novel antidepressants.
Long-term stress is the main risk factor for MDD. It can induce dysfunction in the hypothalamic-pituitary-adrenal (HPA) axis, which is also related to MDD etiology4,5. As described previously, the HPA axis plays a critical role in stress-induced psychiatric pathophysiology including depression and anxiety disorders by increasing corticosterone levels6,7,8,9. Many animal models have been based on sustained activation of the HPA axis, which is observed in patients with MDD4. Moreover, high glucocorticoids induced by chronic stress and subcutaneously&#160;injected glucocorticoids cause depressive behaviors along with neural cell death, atrophy of neuronal processes, and reduced adult neurogenesis in the brain of rodents10,11. Another important brain area associated with depression is the medial prefrontal cortex (mPFC). The mPFC plays a crucial role in controlling brain subregions, such as the hypothalamus and amygdala, that control emotional behavior and stress responses8,9. For instance, lesions in the dorsal mPFC induced HPA axis dysfunction and enhanced corticosterone secretion due to restraint stress12,13. A recent study also showed that repeated restraint stress increased corticosterone levels, which could be decreased by glutamine supplementation via glutamate-glutamine cycle between neurons and astrocyte in the mPFC9.
The first chronic stress paradigm used to study the etiology of MDD was suggested by Katz14. Willner et al. then proposed a chronic mild stress (CMS) model based on the findings of Katz. They confirmed that the model had predictive validity by observing that antidepressants restored CMS-induced anhedonic-like behavior15,16. Typically, the CMS model consists of a combination of various mild stresses, such as mild noise, cage tilting, wet bedding, altered light-dark cycles, cage shaking, forced swimming, and social defeat. The CMS model is widely utilized by researchers; however, this model is of poor replicability, and time- and energy-inefficient. Therefore, there is a growing demand for a standardized and simplified protocol for induction of depressive-like behavior and physiological analysis to evaluate depression. Compared to the CMS model, the chronic immobilization stress (CIS; also known as chronic restraint stress) model is simpler and more efficient; therefore, the CIS model can be widely used in chronic stress studies17,18,19,20,21,22,23,24. In addition, CIS can be used in both male and female mice to develop depressive behaviors25,26. During CIS, animals are placed in a body-fit sized cylinder for 1-8 hours per day for 2 or 4 weeks9,27,28. Of these, restraint stress condition for 2 hours per day for 2 weeks is sufficient to cause depressive behaviors with minimal pain in mice9,28. Under restraint conditions, blood corticosterone levels were rapidly increased9,28,29. Several studies have shown that the CIS model has predictive validity, confirming that CIS-induced depressive-like symptoms are restored by antidepressants19,20,30,31. Herein, we report the detailed procedures of CIS, as well as some behavioral and physiological outcomes after CIS in mice.

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			<td>1 ml disposable syringes</td>
			<td>Sungshim Medical</td>
			<td>P000CFDO</td>
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			<td>Balance</td>
			<td>A&#38;D Company</td>
			<td>FX-2000i</td>
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			<td>Ball nozzle</td>
			<td>Jeung Do B&#38;P</td>
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			<td>CCTV camera</td>
			<td>KOCOM</td>
			<td>KCB-381</td>
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			<td>Corticosterone ELISA kits</td>
			<td>Cayman Chemical</td>
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			<td>Digital lux meter</td>
			<td>TES</td>
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			<td>Ethovision XT 7.1</td>
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			<td>Isoflurane</td>
			<td>HANA PHARM CO., LTD.</td>
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			<td>Mice</td>
			<td>Koatech</td>
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			<td>C57BL/6 strain</td>
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			<td>Restrainer</td>
			<td>Dae-jong Instrument Industry</td>
			<td>DJ-428</td>
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			<td>Saccharose (sucrose)</td>
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			<td>Small animal isoflurane anaesthetic system</td>
			<td>Summit</td>
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			<td>Acrylic bar</td>
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			<td>The apparatus was made in the lab for TST test</td>
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			<td>Tail suspension box</td>
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			<td>The apparatus was made in the lab</td>
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			<td>Timer</td>
			<td>Electronics Tomorrow</td>
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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.

In the representative experiment, all data were acquired from 6 - 8 mice per group. Representative materials and the method to insert the mouse voluntarily into the restrainer are shown in Figure 1.
To perform the behavioral test and blood sampling after CIS induction, mice were subjected to the experimental procedure as summarized in Figure 2A. As shown in Figure 2 and Figure 3</stro...

Watch this Scientific Journal Video about A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice at JoVE.com

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

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, ...

Begin by setting the room to light at 200 lux using a digital lux meter. House the mouse in a separate cage at least a week before testing, and place the mouse in the testing room for at least 30 minutes before the experiment. Gently hold the mouse tail to avoid tensing the mouse, and then carefully place it on a rough surface.Cover the restrainer with a small white towel and then gently place the mouse at the opening of the restrainer, so that the mouse enters the restrainer voluntarily. Next, place the closure to restrain the mouse as tight as possible, being careful to avoid damage to the body, such as tail, feet, and testicles. Lastly, during the exposure to the restrainer, measure body weight and food intake every 48 hours.Prior to testing, habituate the mice to the presence of two drinking bottles, one containing 0.1 molar sucrose and the other containing plain water for 48 hours. Switch the positions of the two bottles after 24 hours to reduce any confounding produced by a side bias. On the third day deprive the mice of water for 24 hours.Then on the day of the experiment expose the mice to two drinking bottles for six hours. After three hours, switch the position of the water bottles. Record the volume in milliliters of sucrose solution and water consumed, and then calculate the animal's affinity to sucrose.Finally, calculate sucrose preference as a percentage of the volume of sucrose consumption over the total fluid consumption during the test. Begin by bringing the chronic restraint stress induced mice into the testing room at least 30 minutes before beginning the tail suspension test. Set the room light to dim conditions at 50 lux.Next, place the camera about 40 centimeters from the mouse. Suspend the mouse firmly from the horizontal bar using adhesive tape. After the mouse is positioned in the middle of the suspension box start recording, and observe the behavioral alterations continuously for six minutes.If the mouse attempts to climb its tail use a stick or climb stopper to prevent it from doing so. At the end of the experiment move the mouse to its home cage and carefully remove the tape from its tail. Finally, use video tracking software to analyze the accumulated time of immobile periods.Results indicate decrease in body weight for the CIS group and significantly less food intake at day four. Compared to controls, a decrease was shown for sucrose preference in the CIS group, while there was a significant increase in immobility time and corticosterone. Further, a glutamine supplemented diet ameliorates depressive like behaviors, as shown for body weight, food intake, sucrose preference, immobility, and corticosterone.

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Research

JoVE Journal

Behavior

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 ...

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

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

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

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

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

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

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

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

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

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

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 ...

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 ...

Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...