The mouse model of major depressive disorder induced by chronic unpredictable mild stress

In order to examine neuron-specific mechanisms underlying resilience versus susceptibility to chronic stress, we applied C57 GAD-GFP mice whose GABAergic neurons were genetically labeled by green fluorescent protein (GFP) [59]. Male mice were used starting at postnatal day 21. In week one for their adaptation to the experiments, their body weight, locomotion, sucrose preference and Y-maze tests were measured to collect self-control data. The mice of showing consistent value in these measurements at postnatal day 28 were separated into two groups, chronic unpredictable mild stress (CUMS) and control, in order to reduce the variations among these mice. The control mice lived without the following stresses. The use of juvenile mice to examine the occurrence of major depression versus resilience is based on a fact that young individuals have high prevalence to suffer from major depression in response to chronic stress [46].

According to depression risk factors, such as weaknesses in cognitive function, emotional regulation, social interaction skill, circadian and stress response [21], we applied the CUMS to the mice in the following principle. The mice lived in stress environments, made efforts to challenge these stressful conditions and experienced defeat outcomes, which drove them to feel cognitive and emotional inabilities, and in turn to fall into anhedonia and low self-esteem. The protocols for the mice experiencing the CUMS included social isolation, tilted cage, empty cage, damp sawdust cage, restraint space, white noise, strobe light and circadian disturbance [37, 46, 67]. Except for social isolations, these conditions were randomly selected to treat the mice in the manners of their separations or combinations every day. These treatments were applied about 1~14 hours in durations and 1~12 hours in intervals. The durations and intervals were unpredictable to these mice (Table ​(Table11 in [46]). This CUMS was sustained for three weeks until some mice expressed anhedonia and low self-esteem. We did not use extreme stress in a single pattern, such as learned hopelessness, electrical shock, social defeat and tail clamp, since these protocols might induce the outcome similar to anxiety, such as posttraumatic stressful disorder [46].

Whether the CUMS-treated mice in three weeks fell into anhedonia and low self-esteem was tested in day 29~31. The sucrose preference test (SPT) was used to evaluate anhedonia, the Y-maze test (YMT) were used to assess the loss of interest to their partners and the forced swimming test (FST) was used to estimate their self-esteem [16, 46, 68–71]. The SPT was done by 1% sucrose water versus water for four hours, whose value was presented as the ratio of the ingested sucrose water to the ingested sucrose water plus pure water. The YMT was performed by monitoring mouse staying in a special arm and other two arms for 2 minutes. The end of this special arm included a female mouse (named as M-arm). M-arm stay time was presented by the ratio of stay time in M-arm to that in three arms. The FST was conducted by recording immobile time in a water cylinder (10 centimeters in diameters and 19 centimeters in water depth at 25 ± 1°C). In the quantification of the FST, immobile time was measured. In these tests, the SPT was given once a week, the YMT was given before and after the CUMS, and the FST was given one time after the CUMS. Before the SPT, the mice in groups of CUMS and control were deprived from food and water for 3 hours to drive their intension of drinking water. In the YMT, these arms were cleaned by 70% ethanol and then water after each test to reduce the effect of odor on the test. Carefulness in these tests was taken by performing them in a quiet room, no addition stresses, same circadian circle for all mice and their adaptation in the test environment.

Depression-like behaviors were accepted if the mice in the CUMS group showed the decreases in sucrose preference (twice at ends of week two and three) and M-maze stay time, as well as the increase in immobile time, compared to these values during their self-control period (week one for adaption) and in control group mice. These measurements in each of mice would be considered as significant change if the values of the SPT and YMT reduced above 10% of its self-control values and the value of the FST increased above 10% values from control group mice. These standards set up based on the averaged values in our previous studies [37, 46]. The mice with significant changes in all of these three tests were defined as CUMS-induced depression-like mice or depression-like mice, and those with no changes in these three tests were named as resilience mice. As showed in Table ​Table1,1, CUMS-treated mice in 3 weeks met this criterion about 28% (i.e., their vulnerability to chronic stress), and 22% of them were resilience (i.e., their invulnerability to chronic stress). These depression and resilience mice were used for the study of electrophysiology. As 30% of CUMS-treated mice met the depression criteria and all CUMS-treated mice did not show a change of the SPT at the end of week one, stressful situations in our study were thought to be mild stress. The mice that met the significant changes in one and/or two measurements are atypical in major depression, called as atypical depression.