4.5. Anxiety Model

A total of 104 Sprague-Dawley rats, aged 60 days and weighing 150–170 g, were purchased from Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China, Certificate no.: SCXK-2016-0006). The first day of arrival, the rats were individually caged in a controlled environment (22 ± 2 °C; 50% RH) and were maintained on a 12:12 h light/dark cycle (light phase: 07:00–19:00). They were also allowed to eat and drink freely. Rats were habituated to the laboratory environment for 1 week and gently handled each day. Behavior experiments were performed between 09:00 and 15:00. The experimental procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Psychology of the Chinese Academy of Sciences and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

The methods and dosages used in this study are all referred to in our previous studies and some adjustments were made [19]. Diazepam at the dose of 1 mg/kg was given in the DZP group. The dosage of ZZX in each group was 1.2 g/kg (Table 6). The control group and empty bottle stress model group (EBS group) were given physiological saline as placebo. All drugs mentioned were dissolved in physiological saline and prepared before use. The volume of administration was 1 mL/100 g of body weight. All rats were given drugs orally once a day for 7 days (Figure 11). Behavioral tests were conducted 1 h after the last administration. After the completion of behavioral tests, rats were decapitated immediately to obtain brain and blood samples.

The flow chart of animal experiments.

The EBS protocol was performed following the procedure described in the previous study, and simple modifications had been made [19,56] (Figure 11). A total of 21 days were taken to conduct the empty bottle stimulation anxiety model. For the first day after 1 week of acclimatization (day 7), except for the control group, rats in other groups were trained to drink water at 09:00 h to 09:10 h and 21:00 h to 21:10 h by allowing them access to water bottles only during these time periods for 1 week. After 1 week of training to drink water at fixation time (day 14), rats in the EBS group; DZP group; ZZX1-10 groups were randomly given empty bottles during one of the two watering periods for 2 weeks to induce anxious emotion. There was no restriction on the rats in the control group during all the time (Table 6).