Sex differences in peripheral monoamine transmitter and related hormone levels in chronic stress mice with a depression-like phenotype

Mice

Sixty C57BL/6 mice (male/female) of 3–4 weeks weighing 11 g–18 g which were healthy SPF mice without gene modification were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. (License no.: SCXK (Shanghai) 2013-0006), and housed in a temperature-controlled room (22 ± 2 °C) and humidity-controlled (55 ± 5%) on a 12 h/12 h light/dark cycle with access to standard chow diet and water ad libitum. After 1 week of acclimatization, mice with same gender were randomly divided into two groups (n = 15): non-treated control group (fed normally without any stress) and chronic stress group (exposed to 8 weeks of chronic unpredictable mild stress). We selected random number generator to generate random seed number for grouping. Animal procedures were complied with the ARRIVE guidelines and performed in accordance with the guidelines of the U.K. The Animal (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. The animal experiment protocol was approved by the Bioethics Committee of Fengxian Hospital, Southern Medical University (approval number: 22010737).

Chronic stress model

Chronic unpredictable mild stress (CUMS) was a classical depression-like behavior/phenotype animal model. Mice were exposed to a series of stressors over several week to induce a range of behavioral alterations including apathy and anhedonia. Those behavioral changes fairly simulate the behavioral characteristics of patients with clinical depression (Katz, 1982; Willner et al., 1987). Male/female mice in the chronic stress group were housed individually and exposed to 8 weeks of chronic unpredictable mild stress (CUMS) once a day as previously described (Zeng et al., 2014). The same stressor was not allowed to use consecutively. Mice were moved to an isolated room when exposed to stress, and moved back after stress treatment. Meanwhile, male/female mice in the control group were raised in the same experimental environment without any stress. The subjected chronic stressor patterns are shown in Table 1.

Behavior assessment

Depression-like phenotypes were assessed with the open field test (OFT) (Walsh & Cummins, 1976) and sucrose preference test (SPT) (Willner et al., 1987). The OFT of each mouse was conducted in a quiet room with Animal Behavior Trajectory Video Analysis System (Labmaze, ZSDC Science Technology Co., Ltd., China) after CUMS model establishment. Mouse was placed individually in the center of the animal behavior track analysis instrument (100 cm × 100 cm × 40 cm) which were divided into 25 grids (20 × 20 cm²). The number of squares crossed by each mouse within 5 min was recorded as its locomotion activity scores, and the number of standing times of each mouse was recorded as its exploratory behavior scores.

The sucrose preference test (SPT) procedure was also performed after CUMS model establishment. Briefly, mice were individually exposed to 1% sucrose solution (w/v) for the first 24 h. Then one bottle of 1% sucrose solution and another bottle of tap water were presented at random positions to mice for the next 24 h, followed by food and water deprivation for the third 24 h. On day 4, two bottles were weighed and presented to each mouse for 4 h. The position of the two bottles was set randomly. The sucrose preference was calculated by the equation as follow: Sucrose preference = sucrose consumption (g)/(water consumption (g) + sucrose consumption (g)) ×100%.

Peripheral blood sample collection and determination

Before and after CUMS model establishment and behavioral assessment, mice were kept unconscious by intraperitoneal injection of 1% sodium pentobarbital 0.1 ml/20 g. The orbital venous plexus blood of mice was collected by glass capillary at 8:00 am. All blood samples were placed in a coagulation tube and allowed to clot for 2 h at room temperature before 1000 × g for 15 min. Then the upper serum was collected for assaying the level of 5-HT, dopamine, norepinephrine, androgen, estrogen, oxytocin and prolactin, adrenaline, corticosterone and ACTH. The ELISA kits of 5-HT, dopamine, norepinephrine, androgen, estrogen, oxytocin and prolactin, adrenaline, corticosterone, and ACTH were purchased from Shanghai Jianglai Biological Technology Co., Ltd (Shanghai, China). The ELISA test was carried out according to the operation instructions of the kit, as follows:

(1) Dilute the antibody to 1–10 µg/mL with buffer solution. Add 0.1 mL to the reaction well overnight at 4 °C. The next day, discard the solution in the hole and wash it three times with washing buffer.

(2) Add sample: add 0.05 mL diluted sample to the coated reaction well and incubate at 37 °C for 1 h. Then wash (blank well, negative control well and positive control well at the same time).

(3) Add enzyme-labeled antibody: add 0.05 mL of freshly diluted enzyme-labeled antibody (titrated dilution) into each reaction well. Incubate at 37 °C for 0.5–1 h, then wash for three times.

(4) Add substrate solution for color: add 0.1mL TMB substrate solution temporarily prepared in each reaction well, 37 °C for 10–30 min.

(5) Stop the reaction: add 2M sulfuric acid 0.05 mL into each reaction well.

(6) Results: on a white background, directly observe the results with naked eyes: the darker the color of the reaction hole, the stronger the positive degree, the negative reaction is colorless or very light, according to the depth of the color, to “+”, “-” sign. The OD value can also be measured: on the ELISA tester, at 450 nm, the blank control well is zeroed and then the OD value of each well is measured. If the OD value is greater than 2.1 times of the specified negative control, it is considered as positive.

Data statistical analysis

Data statistical analysis were carried out using SPSS 25.0. Data is presented as mean ± SD. Homogeneity of variance test and normal distribution test were used to evaluate whether the data met the assumptions. Two-way ANOVA were used to analyze the changes of transmitter and hormone levels between two gender mice before and after chronic stress treatment. All analyses were made with a 95% CI, and the level of significance was set at p < 0.05. P values were ^∗p < 0.05, ^∗∗p < 0.01.

Verification of chronic stress-induced depression-like phenotype

Behavior assessments confirmed that the chronic stress-induced depression-like phenotype was established successfully. As shown in Figs. 1A & 1B, locomotion activity scores and exploratory behavior scores in stress mice were significantly decreased compared to control group after stress model establishment (p < 0.05). Further analysis demonstrated that sex difference was found: the locomotion activity scores in male stress mice declined more than in female stress mice (18.76% vs 7.82%, n = 15), while the exploratory behavior scores in female stress mice declined more than in male stress mice (35.86% vs 46.79%, n = 15). There was an interaction between gender and stress intervention on the effect of locomotion activity scores (p = 0.012).The sucrose preference percentage of two groups both declined , further analysis displayed that there was a sex difference of the decline range for male and female stress mice respectively (21.91% vs 14.24%, n = 15), as shown in Fig. 1C.

Sex difference in monoamine transmitter after model establishment

After 8 weeks of chronic stress model establishment, monoamine transmitters such as 5-HT, dopamine, and norepinephrine in serum decreased in chronic stress group mice as compared to the baseline. There was an interaction between gender and stress intervention on the effect of 5-HT (p = 0.001) and norepinephrine (p = 0.011). As shown in Figs. 2A, 2B, the level of 5-HT in male stress mice decreased more than that in female stress mice (41.19% vs 27.23%, n = 12), while the level of norepinephrine in male stressed mice decreased less than that in female stress mice (38.83% vs 59.17%, n = 12). As for dopamine level, the reduction was slightly in male and female mice (9.51% vs 9.78%, n = 12) but there was no statistical significance of dopamine between the male and female mice (p = 0.837), as shown in Fig. 2C.

Sex difference in sex hormone after model establishment

After stress model establishment, sex hormones such as androgen, estrogen, oxytocin, and prolactin in serum were significantly decreased in chronic stress group mice compared with baseline. Interaction between gender and stress intervention existed in estrogen (p = 0.004), oxytocin (p = 0.043) and prolactin (p = 0.004). There was no an interaction between gender and stress intervention on the effect on androgen (p = o.332). Androgen (Fig. 3A), estrogen (Fig. 3C), and prolactin (Fig. 3D) levels in male stress mice decline less than those in female stress mice (13.42% vs 32.64%, 45.75% vs 69.32%, and 16.12% vs 23.14%, respectively, n = 10). Meanwhile the oxytocin (Fig. 3B) in male stress mice decreased more than in female stress mice (33.11% vs 17.11%, n = 10).

Sex differences in stress hormone after stress model establishment

After stress model establishment, adrenaline, corticosterone, and ACTH were significantly increased in chronic stress group mice compared with baseline, whereas there was no interaction between gender and stress intervention. As shown in Fig. 4, serum adrenaline (Fig. 4A) in male mice increased more than in female mice (p = 0.765) (24.42% vs 22.22%, n = 10) and ACTH (Fig. 4C) in both male and female mice (p = 0.914) increased similarly (51.96% vs 51.81%, n = 10). However, the increasing amplitude of corticosterone (Fig. 4B) in male stress mice was more than that in female stress mice (p = 0.156) (38.46% vs 21.80%, n = 12).

Sample size

According to literature and experimental results, behavioral scores of subjects were used as indicators to determine sample size in hypothesis testing. The mean behavioral scores of the control group were 173.14 ± 21.84, and the behavioral scores of the expected stress group decreased by 25 points, establishing a bilateral alpha is 0.05 and a confidence interval of 90%. According to the sample size calculation formula, the control group and the experimental group each need 15 mice.

Study limitations

In this study, we only used a single species of C57BL/6 mice model to conduct the research. Due to the differences between different species, our results may not be universal. The changes of the transmitters and hormones displayed in mice model may not exactly extrapolated in human beings.

Statement

The protocol was prepared before the study and was not registered.