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
<ol>
	<li>Mice
	<ol>
		<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.

<table>
	<tbody>
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			<td>1 ml disposable syringes</td>
			<td>Sungshim Medical</td>
			<td>P000CFDO</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>A&#38;D Company</td>
			<td>FX-2000i</td>
			<td></td>
		</tr>
		<tr>
			<td>Ball nozzle</td>
			<td>Jeung Do B&#38;P</td>
			<td>JD-C-88</td>
			<td></td>
		</tr>
		<tr>
			<td>CCTV camera</td>
			<td>KOCOM</td>
			<td>KCB-381</td>
			<td></td>
		</tr>
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			<td>Corticosterone ELISA kits</td>
			<td>Cayman Chemical</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Digital lux meter</td>
			<td>TES</td>
			<td>TES-1330A</td>
			<td></td>
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			<td>Ethovision XT 7.1</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td></td>
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			<td>Isoflurane</td>
			<td>HANA PHARM CO., LTD.</td>
			<td></td>
			<td>Ifran solution</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Koatech</td>
			<td></td>
			<td>C57BL/6 strain</td>
		</tr>
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			<td>Restrainer</td>
			<td>Dae-jong Instrument Industry</td>
			<td>DJ-428</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose (sucrose)</td>
			<td>DAEJUNG</td>
			<td>7501-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal isoflurane anaesthetic system</td>
			<td>Summit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic bar</td>
			<td></td>
			<td></td>
			<td>The apparatus was made in the lab for TST test</td>
		</tr>
		<tr>
			<td>Tail suspension box</td>
			<td></td>
			<td></td>
			<td>The apparatus was made in the lab</td>
		</tr>
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			<td>Timer</td>
			<td>Electronics Tomorrow</td>
			<td>TL-2530</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bottle</td>
			<td>Jeung Do B&#38;P</td>
			<td>JD-C-79</td>
			<td></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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