Neurotransmitter and behavioral changes in brain regions of mice induced by restraint stress and effects of exercise intervention
Abstract: To investigate the effects of restraint stress on neurotransmitters and behavior in mice and the effects of aerobic exercise. Eighty-one C57BL/6 mice of 1 month old were randomly divided into 4 groups: control group (Control, n=20), restraint stress group (Stress, n=20), exercise group (Ex, n=20). Only), exercise restraint stress group (Stress+Ex, n=20). The control group was quietly reared, and the restraint stress group was subjected to 2 weeks of restraint stress. The exercise group performed 2 hours of treadmill exercise every day, and the exercise restraint stress group simultaneously performed restraint stress and treadmill exercise. The results showed that in the behavioral test, the social behavior of the restraint stress group decreased significantly, the anxiety and depression behavior levels increased significantly (P<0.05), and exercise intervention could alleviate the effect of restraint stress on these behaviors. Biochemical indicators also indicate that aerobic exercise effectively relieves the behavior caused by restraint stress and changes in monoamine neurotransmitters. The results show that restraint stress can lead to changes in social and emotional related behaviors, and exercise intervention can play an effective mitigation role.
Key words: exercise physiology; restraint stress; exercise; social behavior; single neurotransmitter; mouse
Continued stress can cause people or animals to change their social habits, reduce their cognitive ability, and even develop emotional disorders such as depression or anxiety. Therefore, exploring the mechanism of stress stress on the brain and behavior of individuals is of great significance to the protection of human health. Binding stress is one of the widely used animal models that simulate stress stress in human life. Restraint stress affects a variety of behavioral and biological indicators in animals, including cognitive impairments in learning and memory, and changes in social interaction behavior. Acute restraint stress can cause negative emotions in the body, causing damage to hippocampal neurons in the brain, increased dendritic spines and increased growth. Chronic restraint stress causes behavioral changes due to changes in the sensitivity of the HPA axis. A large number of studies have found that chronic restraint stress can significantly reduce the cognitive function of experimental animals with changes in the levels of catecholamine neurotransmitters and metabolites in the cerebral cortex, hippocampus and peripheral blood. Restraint stress causes neuroendocrine changes, including a decrease in catecholamines and a decrease in the sensitivity of corticosterone stress response. This neuroendocrine alteration is associated with increased function and expression of serotonin receptors in neurons and changes in serotonin neurotransmitter secretion. Chronic stress can make neurons in the amygdala extremely active, causing damage to the corresponding neurons. Animal studies have shown that exercise can regulate a variety of neurotransmitter systems, such as serotonin (5-HT), dopamine (DA), norepinephrine (NE) and their metabolites, which in turn can play a positive role in intervention.
Although studies on the effects of restraint stress on neurotransmitters and behaviors have been reported, there are relatively few studies on the effects and mechanisms of exercise on chronic restraint stress. Therefore, this study used the mouse chronic restraint stress model to investigate the effects of exercise intervention and explore its mechanism of action.
1 Experimental materials and methods
1.1 Reagents and instruments
1) Norepinephrine (NE), dopamine (DA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) were purchased from Sigma, USA, purity ≥ 98.0%; acetonitrile: chromatographically pure According to Fisher Scientific Company of the United States; perchloric acid, potassium bicarbonate, glacial acetic acid, etc. are all analytically pure, Nanjing Chemical Reagent Co., Ltd. products; experimental water is homemade secondary re-distilled water.
2) LC.10AD vp high performance liquid chromatograph, SPD-10AD vp fluorescence detector, CTS column for Shimadzu Shim-pack VP-ODS (150 ibm × 4.6 mm × 5 μm) (Japan Island)津.) GL.88B vortex mixer (Jiangsu Haimen Kirin Medical Instrument Factory); electric constant temperature water bath device (Jiangsu Guangdu Electromechanical Equipment Co., Ltd.); Supermaze animal behavior analysis system, social behavior box, elevated cross maze, new object recognition Box, forced swimming experiment box (Shanghai Xinsoft Company) .
1.2 Experimental animals and experimental design
C57BL/6 mice (3 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The mice were housed in a ventilated cage at room temperature of 20 to 25 ° C, relative humidity of 60%, and a light-dark cycle of 12 h, kept quiet, and food and water were provided on time. All mice were pre-adapted for 1 week prior to the formal experiment and the mice were weighed daily. Eighty mice were randomly divided into 4 groups, 20 in each group, control group (n=20), restraint stress group (n=20), exercise group (n=20), exercise restraint stress group (n =20 only). The specific implementation scheme is as follows: adaptive feeding for 1 week, the restraint stress is 120 minutes starting every day at 10:00 am, the running time for the treadmill is 18:00 for 30 minutes, and training is 5 days per week for 2 weeks. Restraint stress method: Take a 50 m L cylindrical centrifuge tube (with a 3 mm radius hole at the bottom of the tube), place the mouse in a tube that can only hold the entire body of the mouse, and place the tube in different cages in the same room. In the middle, the placement time is from 9:00 am to 11:00 am. Treadmill exercise method: the treadmill movement on the flat slope, the speed is 0.8 km/h, the slope is 0°, and the mice are subjected to a 15-minute adaptive training at 18:00 on the 1st day before the start of the experiment, and then the second day is pressed. The above exercise program training. Among them, the restraint stress group and the restraint stress and exercise intervention at the same time; the restraint stress group only restrained the stress; the exercise group only exercised the exercise; the control group was fed with other groups of mice, no exercise intervention and restraint stress. All animal experiments were performed in accordance with the guidance documents of the Ethics Committee of Southeast University.
1.3 Indicator detection
1 month old mice are in adolescence, in order to study whether restraint stress can lead to changes in anxiety, depression and social behavior. After two weeks of experimentation, social behavior experiments, elevated plus maze, novelty-inhibition feeding trials, and forced swimming experiments were performed to verify the effects of anxiety and depression levels and exercise intervention on these behaviors in mice. After 24 hours of equilibration, the rats were sacrificed and the levels of monoamine neurotransmitters in different brain regions were quickly detected.
1) Social behavior experiment.
The social behavior experiment used a social behavior observation room containing 3 rooms of the same size (40 cm × 40 cm × 20 cm), with an empty room in the middle and a passage leading to the rooms on both sides. Squirrel cage. There are two types of experimental mice, one is a stranger for social behavior and the other is an experimental mouse. The test used 2 social behavior-specific strange mice 1 and 2, all of which were male, similar in age and weight, and had the same strains as the experimental mice and had not been exposed before the experiment. Separate the two rooms on the left and right sides with an opaque plexiglass plate. Before the start of the experiment, the rats were placed in the middle room of the behavior observation box for 5 min, during which the strange mice were randomly placed in the squirrel cage of one room. Experimental mice can sneak into each other with strange mice, but they are unable to make physical contact. At the beginning of the formal experiment, the isolation plate was removed, and the interaction time and room stay time of the experimental mice with the empty mouse cage or the strange mouse were recorded by the animal behavior analysis system. Interaction time = experimental mice were directly exposed to squirrel cages + erect sniffing time within 2 cm. The residence time of the experimental mice in each room was based on the head and limbs of each mouse entering the room. After the experiment, the experimental mice were taken out and returned to the cage. After each experiment, the entire test set was cleaned with a volume fraction of 70% alcohol for the next experiment. The specific process is shown in Figure 1.
As shown in Figure 1, the cage is a strange mouse, and the extracellular cage is the experimental mouse. The social behavior experiment consists of two stages: the first stage is simultaneously recorded (A) the experimental mouse in the strange mouse 1 or the empty mouse The time of stay in the room where the cage is located and (B) the time at which the experimental mouse interacts with the empty mouse cage or the strange mouse 1; the second stage is the (C) stay of the experimental mouse in the room of the strange mouse 1 or the strange mouse 2 Time and (D) experimental mice interacted with strange mice 1 and strange mice 2 .
2) The elevated cross maze experiment.
Place the mouse in the middle of the cross maze, 1 m above the ground, with closed arms on both sides and open arms on the other two sides. The time and path of the mouse staying in the open and closed arms was recorded using an electronic path system. The labyrinth is washed with 70% ethanol and water, dried and tasteless, and the next animal experiment is carried out.
3) Novelty - inhibition of feeding trials.
After the mice were deprived of the diet for a while, they were placed in a new object identification box (60 cm x 60 cm x 30 cm). One spherical mouse grain was placed in the center of the bottom of the experiment box, and each mouse was placed in the corner of the bottom of the experiment box and freely moved for 12 minutes. The time of the first time each mouse was eaten was recorded, and the diet of each mouse in the cage was recorded 30 minutes after the end of the test.
4) Forced swimming experiment.
Place the mouse in the center of the bucket (water temperature 24~25 °C; bucket radius 20 cm, height 40 cm) for 10 min. The mouse could not touch the bottom of the bucket. The behavior of the mice is automatically recorded by the video path monitoring system. The instrument automatically records the time of the mouse's potential immobility and the last 4 minutes of struggling time (strong limbs struggling and scratching the wall of the bucket).
5) The mice were taken.
Each mouse was weighed and randomized. The body weight of each group was weighed using an electronic balance (JZC-XXC, Shanghai Yajin Technology Co., Ltd.) before the exercise every Monday at 18:00. After the behavior test, the patient was allowed to stand for 24 h, the cervical vertebra was sacrificed, the different brain regions were quickly stripped and blood was taken, and the brain tissue samples were weighed. 50 μL of ice-cold 0.1 mol/L perchloric acid (containing 10-7 mol/L) was added. Vitamin C) is homogenized with a micro-homogenizer, and the homogenate is centrifuged (4 ° C) for 5 min at 5 200 × g, then filtered through a 0.22 μm filter and 10 μL of the filtrate is diluted to 1 m L with 990 μL of distilled water to absorb 20 The μL dilution was injected into the chromatograph for analysis and the other samples were quickly stored at -80 °C.
6) Detection of monoamine neurotransmitters and their metabolites.
Monoamine neurotransmitters and their derivatives in the mouse brain were detected by high performance liquid chromatography with ESA MD-150 column (150 mm × 3.2 mm, 2.0 μm), C18 precolumn (10 mm × 3 mm) , 3 μm). The excitation wavelength is 345 nm and the emission wavelength is 480 nm. The mobile phase is selected: filtered with a 0.45 μm microporous membrane, ultrasonically degassed acetonitrile and 15 mmol/L acetate buffer (p H4.7; 35..65, Volume ratio) Flow rate: 1.0 m L/min, column temperature: 30 °C, injection volume: 20 μL.
7) Data statistics.
All data were entered into SPSS 18.1 statistical software, and the effects of restraint stress and exercise intervention were statistically analyzed by analysis of variance.
3 Discussion
Binding stress is the most common animal model that mimics human life stresses and changes in human social, emotional, and cognitive behaviors. At the same time, restraint stress is also a widely used animal model of stress disorder, which can not only simulate immune decline, cognitive decline, but also cause anxiety and depression in mice. There is increasing evidence that restraint stress has a shaping effect on the brain, which can be manifested as changes in genes, neuroendocrine and behavioral. Stress activates the monoamine transmitter system and affects the structure of the neural network. Therefore, repeated stress can make the body adapt to stress.
Under stress, the 5-HT update rate may increase in the brain of experimental animals, causing excessive hyper- or excessive function of the HPA axis, which in turn leads to a change in 5-HT concentration. Acute stress significantly increased the levels of 5-HT and its metabolite 5-HIAA in the cortex, hippocampus, and amygdala; the chronic stress cortex 5HT and 5-HIAA gradually recovered to or below the pre-stress level. The levels of 5-HT in the brain increased after 3 h of acute exercise or 8 weeks of treadmill exercise. Microdialysis studies found that 5-HT levels in the hippocampus of rats increased after 60 minutes of treadmill exercise. In addition, studies have shown that the level of excitability in rats is positively correlated with the release of DA in the brain, and the level of DA in the hippocampus of animals is decreased after repeated stress. Exercise increased DA levels and metabolite levels in the prefrontal cortex, hippocampus, and striatum of rats. Therefore, previous studies seem to indicate that hippocampal neurons are associated with emotional excitement and inhibition of balance.
In this study, the detection of monoamine transmitters in different brain regions of the stress group showed that 5-HT, DA and NE were reduced in most brain regions, and some results were compared with previous results. Consistently, the restraint stress is a stress animal model that is easily replicable. First, this study demonstrates that restraint stress can increase anxiety and depression in mice. Second, the results of this study also found that restraint stress reduced social behavior in mice. Finally, this study found that restraint stress makes mice Most monoamine neurotransmitters decreased in different brain regions. In summary, the results of this study are consistent with the results of the previous restraint stress mouse model, demonstrating that the restraint stress model successfully replicated the model of simulated stress affecting individual behavioral bias.
At the same time, this study found that after restraint stress in mice, there was a decrease in social interaction behavior, an increase in anxiety and depression. These behavioral changes may be associated with inhibitory and excitatory monoamine neurotransmitter disorders in different brain regions. More importantly, exercise intervention can alleviate the changes in anxiety-depression-related behaviors, monoamine neurotransmitters and their metabolite levels after restraint stress. These results suggest that chronic restraint stress with a period of 2 weeks and 2 hours per day may cause moderate anxiety and depression behavior in mice, while exercise intervention reduces this change in restraint stress.
Numerous studies have shown that exercise can be used to alleviate negative emotions and behaviors caused by stress and improve cognitive function. Based on previous studies, this study used exercise interventions to alleviate stress-induced anxiety, depression, and social-related behavioral changes. The results of this study suggest that exercise alleviates a variety of behavioral and biological changes caused by stress. Some of the results are inconsistent with the previous research results and need further study.
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