30 SPF grade 6-week-old male ICR mice, a total of 30 purchased from Beijing Weitong Lihua Company, license number: SCXK (Jing) 2016-0006 and raised in the Experimental Center of Shandong University of Traditional Chinese Medicine. During the experiment, there were 6 animals in each cage, the temperature was maintained at 22˚C ± 2˚C, the humidity was maintained at 50% - 60%, the black and white cycle was 12 hours, and the food was free. Before the experiment, all mice were acclimatized to the experimental environment for 6 days. All animal experiments were reviewed and approved by the ethics committee of Shandong University of Traditional Chinese Medicine.

mCPP (sigma, 125180-5G) was dissolved in saline to make 0.4 mg/mL. Take an appropriate amount of solution and dilute it to the required concentration.

Elevated plus-maze experiment system, Light-dark box experiment system, Open field experiment system, Super Maze analysis system, SuperTST analysis system (Shanghai Xin Ruan Information Technology Co., Ltd.), RotaRod (ugo basile, 47650), Tail Flick (Chengdu Taimeng Technology Co., Ltd, SW-200), Step-down recorder (Jinan Yiyan Technology Development Co., Ltd, YLS-3TB), Tail suspension box (Xmaze, XR-XX203).

After adaptive feeding for a week, 6-week-old male ICR mice were observed for the general conditions of all animals, and the autonomous activity experiment was conducted. According to the results of the autonomous activity experiment and the body weight of the mice, 24 mice were selected for the experiment.

Twenty-four 6-week male ICR mice were randomly divided into four groups by body weight and total motion distance detected by autonomic mobility, namely the control group (C), the mCPP low-dose group (L, 1 mg/kg), the mCPP medium-dose group (M, 2 mg/kg), and the mCPP high-dose group (H, 4 mg/kg), with 6 in each group. A single intraperitoneal injection of mCPP was administered with a volume of 0.1 mL/10g. The control group was given 0.9% sodium chloride injection, and a behavioral test was performed 30 minutes after a single intraperitoneal injection. The specific animal grouping and dosage are shown in the following table.

After adaptive rearing for one week, mice were screened for autonomous activity. Except for the LDB, all behavioral experiments were performed 30 minutes after injected with mCPP. The specific experimental procedure was conducted as follows (Figure 1).

Figure 1.Experimental procedure of a mouse model of anxiety induced by a single intraperitoneal injection of mCPP.

4.1.1. Elevated Plus-Maze

EPM was conducted 30 min after a single intraperitoneal injection of mCPP. In this study, the anxiety state of animals was measured by using the conflicted behavior of exploring the new environment and the fear of hanging open arms. SuperMaze software was used to collect and analyze the behavior of mice. The mice were placed in the center of the central platform with their heads facing the open arms, and the following indicators within 5 minutes after release: 1) Number of open arms entries (OE); 2) Time in open arms (OT); 3) Number of close arms entries (CE); 4) Time in close arms (CT). Based on the above indexes, the following results can be obtained: 1) Entries to open arms/total arms (%): OE/(OE + CE) × 100%; 2) Time in open arms/total arms (%): OT/(OT + CT) × 100%. Animals with lower anxiety levels were more likely to explore the open arm, so in the EPM, the higher the percentage of open arm entry and open arm time, the less anxious the animal was at present.

4.1.2. Light-Dark Box

This experiment was carried out 24h after single intraperitoneal injection of mCPP before the EPM. Use SuperMaze software to collect and analyze the behavior of mice. Mice were put in the light box, pulled out the insert in the middle of the light and dark box. We recorded behavioral indicators of the mouse within 5 minutes. At the end of each test, the device was wiped with 75% alcohol. Observation indexes were: 1) The number of entrances in the light box; 2) Time spent in light box (%): Time spent in light box/total area × 100%. When the mouse is less anxious, the time and frequency of exploring the bright box will be longer. On the contrary, when the mouse was more anxious, it tended to move in the dark box.

4.1.3. Open Field Box

OFT was conducted 30 min after single intraperitoneal injection of mCPP. We used SuperMaze software to collect and analyze the behavior of mice. The open field box (50 cm × 50 cm × 50 cm) was divided into 9 grids on average. Mice were placed in the center grid of the box, and motion state of the mice was tracked and recorded for 5min under normal light. Before the detection of the next mice, the chamber was cleaned with 75% ethanol to completely remove the odor left by the previous mice. The main indicators collected in the experiment are total distance (m), center zone distance (dm), number of entries to central zone. The less the mice enter the central area, the shorter the residence time in the central area, and the shorter the distance to the central area, the more anxiety the mice have. The fewer times the mice entered the central area and the fewer the distances in the central area, the more anxious mice were.

4.1.4. Hole-Board Test

HBT was conducted 30 min after single intraperitoneal injection of mCPP. The instrument is assembled by putting a plastic plate with 16 holes in the box (50 cm × 50 cm × 50 cm). The HBT was based on the nature of mice to explore caves, using novelty and fear to control the animal's behavior under new environment, and avoiding to reflect the effects of these two factors. The movement of the mice was recorded for five minutes, and the chamber. Observe the head-dip counts and head-dip latency of mice. The decrease of head-dip counts and the increase of head-dip latency indicated anxious behavior in mice.

Rotarod tests require animals to maintain balance and continuous movement on a rotating roller. Mice with dyskinesias or poor coordination will fall down faster. The fall time of the mice is statistically analyzed to evaluate the exercise athletic ability of the mice. The shorter the mice persist on the rotarod, the worse the coordination ability of their exercises. The day before the experiment, mice were placed on the rotarod for regular rotarod training. The mice were adapted to the rotarod for 30 seconds. After the mice were stable, the rod rotator was started, and the rotation speed was set at 10 r/min.

The rotarod test was conducted 30 min after a single intraperitoneal injection of mCPP. In the formal experiment, we placed the mice that had undergone conventional rotarod training on the rotarod with a constant speed of 25 r/min, recorded time the mouse stayed on the rotarod, dropped or grasped the rotarod and followed the rotarod to rotate three times all regarded as the end of the experiment. The rotation time was set to 300 seconds to ensure that the difference in the exercise balance ability between the mice was displayed without causing fatigue damage to the mice. Each mouse performed 3 rotarod tests with an interval of 30 minutes each time, the final experimental result was the average of three experiments.

Tail-flick test was conducted 30 min after a single intraperitoneal injection of mCPP. The time of tail-flick response in mice was regarded as the indicator of pain response. The light intensity of the mice tail light pain tester to 50%, and the detection time is 10 s. We put the mice in the mouse holder, placed the mouse fixed tube seat on the top surface of the instrument, and adjusted its position so that the tail tip of the mice on the tail-tip positioning line, exposed the tail for light irradiation. The exposure site was fixed at the middle and lower 1/3 of the tail of each mouse. After the mice were quiet, the pain test was carried out, and the light bulb was lit. When the rat tail swings, the photoelectric switch automatically turns off the light and stops timing. The minimum unit of timing was 0.1 s. The incubation period from the beginning of the irradiation to the appearance of the tail-flick reaction was recorded as the pain threshold. In order to protect the tails of mice from damage, those who did not flick their tails for more than 10 s were recorded as 10 s, and the latency of tail flicks in each group was statistically processed.

Training period: After 30 minutes of intraperitoneal injection, mice (one in each group in order, and 2 mice were detected at the same time) were placed in the jumping platform experimental device to acclimate for 3 min, and all mice were placed on the safety platform before power-on, and then the power supply (voltage 60 - 80 V, current 0.8 - 1.5 mA) was switched on. The mice would flee to the safety platform after receiving the electric shock. It was a wrong reaction for the mouse to jump from the safety platform and touch the copper grid with its two forefeet. The incubation period of jumping off the platform for the first time within 5 min and the number of shocks from jumping off the platform were recorded, which were used as the learning performance of mice.

Test period: The test was conducted at 8:30 the next day. The mice were placed on a safe platform, with a voltage of 60 - 80 V and a current of 0.8 - 1.5 mA. The experiment lasted for 5 min (no need to adapt). The incubation period of the mice in each group jumping off the platform for the first time within 5 minutes and the number of electric shocks received by jumping off the platform was recorded as the memory performance of the mice.

TST was conducted 30 min after single intraperitoneal injection of mCPP. Super TST software was used to collect and analyze the behavior of the mice. The tail of each mouse was attached to the hook in the suspension box with medical tape, and the changes of the movement state of the mice were collected within 5 min. The first 1 min was the adaptation period, and the state of the mice was analyzed after 4 min. The main indexes collected in the experiment are: immobility time and struggle time. Immobility time was main parameter, and the increase of immobility time indicated that the mice had depression-like behavior.

All data were expressed as Mean ± S.D and analyzed by employing the GraphPad 8.0 software. Data between the two groups were compared using Student’s t-test. Comparisons of data from multiple groups against one group were analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The statistical analysis used P < 0.05 as a basis for the level of significance.

5.1.1. Effects of Single Intraperitoneal Injection of mCPP on the Behavior of Mice in Elevated Plus-Maze

After mice were treated with a single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 30 min, the EPM was conducted to detect the anxiety of mice. The results showed (Figure 2) that the time in open arms was significantly shorter (P < 0.05) (Figure 2(B)) and the percentage of time in open arms was significantly lower (P < 0.05) (Figure 2(D)) in mice treated with high-dose mCPP (4 mg/kg) than control mice.

Figure 2.Effects of single intraperitoneal injection of mCPP on the behavior of mice in elevated plus-maze test. Number of open arms entries (A), Time in open arms (s) (B), Entries to open arms/total arms (%) (C), Time in open arms/total arms (%) (D); Mean ± S.D., n = 4 - 6, *P < 0.05, vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

5.1.2. Effects of Single Intraperitoneal Injection of mCPP on the Behavior of Mice in Open Field Box

After the mice were intraperitoneally injected with mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) once for 30 min, the OFT was performed to test the anxiety of mice. The results showed (Figure 3) that the center zone distance was significantly shorter at dose 2 mg/kg (P < 0.05) and 4 mg/kg (P < 0.01) (Figure 3(B)), the number of entries to central zone was significantly lower at dose 2 mg/kg (P < 0.05) and 4 mg/kg (P < 0.01) (Figure 3(C)) in mice treated with mCPP than that of control mice.

5.1.3. Effects of Single Intraperitoneal Injection of mCPP on the Behavior of Mice in Hole-Board Test

After the mice were given a single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 30 min, the HBT was used to observe the anxiety of mice. Comparing with the control group, the significant reduction of the head-dip counts was observed in the mice treated with 1 mg/kg (P < 0.05), 2 mg/kg (P < 0.05) and 4 mg/kg (P < 0.01) m-CPP (Figure 4(A)). The treatment with 2 mg/kg and 4 mg/kg m-CPP significantly increased head-dip latency of the mice (P< 0.05) compared to the control group (Figure 4(B)).

Figure 3.Effects of single intraperitoneal injection of mCPP on the behavior of mice in open field test. Total distance (A), Center zone distance (dm) (B), Number of entries to central zone (C); Mean ± S.D., n = 5 - 6, *P < 0.05, **P < 0.01 vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

Figure 4.Effects of single intraperitoneal injection of mCPP on the behavior of mice in hole-board test. Head-dip counts (A), Head-dip latency (B); Mean ± S.D., n = 4 - 6,vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

5.1.4. Effects of Single Intraperitoneal Injection of mCPP on the Behavior of Mice in Light-Dark Box

After the mice were administrated with a single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 24 h, the LDB was carried out to evaluate the anxiety of mice. The results showed that there was no significant difference for the entry into the light side and the percentage of time in light side between groups treated with mCPP and control group (Figure 5).

After the mice were intraperitoneally injected with mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) once for 30 min, the rotarod test was used to estimate the athletic ability of mice. The results showed that the injected with m-CPP (1 mg/kg, 2 mg/kg, 4 mg/kg) did not affect the rotating time of the mice compared to the control group (Figure 6).

After the mice were treated with single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 30 min, the tail-flick test was used to measure the pain sensation of mice. The results showed that there was no significant difference for the tail flick latency between groups treated with mCPP and control group (Figure 7).

After the mice were administrated with a single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 24 h, the step-down test was conducted to appraise the passive avoidance response ability of mice. The results showed that there was no significant difference for the number of errors and the step-down latency between groups treated with mCPP and control group (Figure 8).

Figure 5.Effects of single intraperitoneal injection of mCPP on the behavior of mice in light-dark box test. The number of entrances in light box (A), Time spent in light box (%) (B); Mean ± S.D., n = 6,vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

Figure 6.Effects of single intraperitoneal injection of mCPP on the behavior of mice in rotarod test. Mean ± S.D., n = 6,vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

Figure 7.Effects of single intraperitoneal injection of mCPP on the behavior of mice in tail-flick test. Mean ± S.D., n = 5 - 6,vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

Figure 8.Effects of single intraperitoneal injection of mCPP on the behavior of mice in step-down test. Number of errors (A), Step-down latency (B); Mean ± S.D., n = 4 - 6,vs Control, one-way ANOVA analysis followed by Dunnett’s post hoc test, Graphad Prism8.0.1 software.

After the mice were given single intraperitoneal injection of mCPP (1 mg/kg, 2 mg/kg, 4 mg/kg) for 30 min, the TST was used to observe the depression behavior of mice. The results showed that there was no significant difference for the immobility time and struggling time between groups treated with mCPP and control group (Figure 9).