Effects and mechanism of long-term high-fat diet on predatory behavior in rats (field test)

[Abstract] Objective: To study the effect and mechanism of long-term high-fat diet on predation behavior in rats. METHODS: Ten 4-week-old female SD rats were randomly divided into control group and high fat group. The control group was fed with conventional feed, and the high-fat group was fed with high-fat diet. After 15 weeks, the open field experiment and predatory behavior were observed. The animals were sacrificed immediately after the behavioral observation. The c-Fos protein was detected by immunohistochemistry in rats. The expression of the anterior cingulate cortex. RESULTS: The predation of non-competitive predation behavior and competitive predation behavior in the high-fat group was lower than that in the control group (P<0.001). The expression of c-Fos protein in the anterior cingulate cortex of the high-fat group was lower than that in the control group. Group (P < 0.001). Conclusion: Long-term high-fat diet can reduce the predation of rats, and the expression of c-Fos protein in the anterior cingulate cortex is decreased and the function of neurons is decreased.

[Key words] high-fat diet; predation behavior; mine experiment; anterior cingulate cortex; c-Fos; rats with the accelerated pace of life, people's diet structure changed, a large number of high-fat food intake, Can bring a series of health problems, such as obesity, cardiovascular disease, metabolic syndrome, diabetes, etc.; can also lead to neuropsychiatric abnormalities, such as damage to the memory function of mice, causing increased activity in mice and Anxiety and depression behavior; can also cause cognitive impairment in mice. There are few studies on the predation behavior of high-fat diets. The predation behavior of rats involves their handling, hoarding and competition with other rats. It is to search for more food while meeting the current needs. Meeting future use is a strategy for adaptation and survival. Therefore, this experiment will observe the effects of long-term high-fat diet on predation behavior of rats, and observe the changes of c-Fos protein expression in the anterior cingulate cortex of rats, and explore the effect and mechanism of high-fat diet on predation behavior of rats.

1 Materials and methods

1.1 Materials

1.1.1 Experimental animals

Ten healthy Sprague-Dawley (SD) rats, 4 weeks old, were provided by the Experimental Animal Center of Central South University, and the body mass entering the laboratory was 70-90 g. General health requirements: hair color is bright, no hair loss, no disease in the limbs and tail, no skew on the neck. All rats were housed in the same animal room 1 week before the start of the experiment to adapt to the environment, and were free to drink and eat. The food was a standard granular feed. The temperature of the animal room is controlled at 20~22 °C, and the ventilation is good.

Ok, keep the natural light-dark biological rhythm.

1.1.2 Feed and main equipment and chemical reagents

The standard granular feed of rats is provided by the Department of Experimental Animal Science of Central South University; the high-fat feed is self-made (the formula is taken from Zhang Tao, etc.: 50% of common feed, 12% of lard, 10% of milk powder, 5% of soybean meal, 2.5% of peanut) , 5% sucrose, vegetable oil 1%, salt 1%); market experiment box and supporting Supermaze software for Shanghai Xinsoft Information Technology Co., Ltd.; Leica frozen slicer for Wuhan Zhongji Scientific Instrument Co., Ltd.; Nikon microscope for Nikon Instrument Co., Ltd.; Paraformaldehyde is a product of Tianjin Komio Chemical Reagent Co., Ltd.; sodium phosphate sodium is a product of International Group Chemical Reagent Co., Ltd.; disodium hydrogen phosphate is a product of Changsha Antai Fine Chemical Industry Co., Ltd.; Guanghua Technology Co., Ltd.; 0.01 mol/L phosphoric acid buffer solution and DAB color developer are products of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; c-Fos primary antibody is American Santa Cruz product, goat serum, secondary antibody and third antibody It is a US Vector Labs product; anhydrous ethanol and xylene are products of Sinopharm Chemical Reagent Co., Ltd.

1.2 Methods

1.2.1 Establishment of a high-fat diet model

Ten female rats were randomly divided into two groups of 5 each. The control group was fed with normal feed for 15 weeks, and the high-fat group was fed with self-made high-fat diet for 15 weeks. All rats were free to ingest and drink during modeling, and feed and water were changed every 2 days.

1.2.2 Behavioral Testing
1.2.2.1 Market experiment

Open field experiment box (length × width × height: 100 cm × 100 cm × 50 cm), the bottom of the box is marked with 25 equal squares (20 cm × 20 cm), and the 16 squares along the four walls are called the outer grid. The remaining nine squares are the central grid. During the experiment, the rats were placed in the positive central grid, and the number of central grids, the total number of passages, and the duration of the central compartment of the rats were recorded and recorded for 5 minutes. The percentage of total time (5 min) in the central compartment was negatively correlated with the anxiety-like mood of the rats. The total number of passes reflects the animal's motor function.

1.2.2.2 Observation of noncompetitive predation behavior

The predation model refers to the method of Li et al.: The observation of animal predation behavior is carried out in a single, quiet, soundproof room with a open field (length × width × height: 150 cm × 150 cm × 50 cm). The bottom and the four walls of the open field are black. Before the predatory behavior of the rats to be tested, it is necessary to diet for 1 week, and a small amount of food is given every day. The amount of water is reduced enough to reduce the body mass to about 80% before; d fasting (not allowed water) for 12 h, observe the day at 4:00 pm, put the rats into the open field, and after 2 hours of adaptation, put a plastic squirrel cage on the side of the open field (length × width × height: 30 Cm × 18 cm × 16 cm), 250 g of standard rat granulated feed was placed on the barbed wire top of the squirrel cage. After 2 h, the remaining feed on the squirrel cage top and the feed scattered in the open field were collected. And weighed separately. The amount of feed scattered in the open field is the predation of rats.

1.2.2.3 Observation of competitive predation behavior

The observation equipment was observed with non-competitive predatory behavior. Before the observation, the fasting (not forbidden water) was observed for 12 h. The rats were placed in the open field at 4:00 pm on the same day. After 2 h, a plastic was placed on the side of the market. The squirrel cage (length × width × height: 30 cm × 18 cm × 16 cm) was placed in a rat cage with the same sex as the rats to be tested, derived from different litters, rats that had never been exposed before. 250 g of standard rat granulated feed was placed on the barbed wire cover of the squirrel cage, and collected separately after 2 h.

The feed scattered in the market and the remaining feed on the top of the squirrel cage were weighed separately. The amount of feed scattered in the open field is the predation of rats.

1.2.3 Immunohistochemical detection of c-Fos protein in rat anterior cingulate cortex. Rats were quickly removed from the non-competitive predation of rats for 1 h 45 min, using 10% oxyaldehyde (40 mg/kg). Rats were quickly anesthetized by intraperitoneal injection. The brain tissue was placed in the fixative solution and fixed overnight. Then, it was placed in 15%, 30% sucrose PBS solution and immersed in sugar to the bottom. The optimal cutting temperature (optimal cutting temperature, The OCT) composite was embedded and placed in a constant-temperature frozen slicer for serial sectioning. The slice thickness was 30 m, and the section containing the anterior cingulate cortex was taken. Immunohistochemical ABC method was used to observe the immunohistochemical staining of c-Fos and Olympus microscopy. Three sections were selected from the control group and the high-fat group, and the positive cells were counted and counted under a 10×10-fold field of view.

1.3 Statistical processing

Data were expressed as mean ± standard deviation (x ± s), statistical analysis was performed using GraphPad Prism 5.0 software, and a t-test was used for comparison between the two groups. The difference was statistically significant at P < 0.05.

2 results

2.1 Behavioral testing

2.1.1 Market experiment

The results of the open field experiment are shown in Figure 1: The percentage of central compartment retention time, the high-fat group (10.43% ± 2.87%) was not significantly different from the control group (12.23% ± 2.79%) (t = 0.4). 5 0 , P = 0 . 6 6 4 7 ) ; the total number of cells crossed, the high fat group (1 2 7 . 10 % ± 3 7 . 8 9 % ) and the control group ( 9 8 . 9 0 % ± 1 7 The difference was not statistically significant (t = 0.076, P = 0.5172).

2.1.2 Predatory behavior

The results of predatory behavior are shown in Figure 2. In the non-competitive predation behavior and competitive predation behavior of the control rats, the predation per 2 h was (84.80±2.77) g, (89.94±1.96) g. . In the high-fat group, in the observation of non-competitive predation behavior and competitive predation behavior, the predation per 2 h was (13.85±1.08) g, (12.25±1.07) g, respectively; the predation decreased compared with the corresponding control group ( Non-competitive predation behavior t = 21.65, P < 0.001; competitive predation behavior t = 32.17, P < 0.001).

2.2 The immunohistochemical detection of c-Fos protein in the anterior cingulate cortex of rats is shown in Figure 3: The number of positive cells in the control group was (88.17%), and the number of positive cells in the high-fat group was (45.00±2.56)/field, the number of positive cells in the control group was higher than that in the high fat group (t=8.868, P<0.001).

3 Discussion

Predatory behavior is a highly integrated model that can be used to explore different issues in psychology, physiology, neurobiology, etc. At the same time, predatory behavior is also an important model of behavioral science theory research, predatory behavior and decision-making, efforts Cognitive functions such as payouts, cost/benefit estimates are closely related. Using predation behavior to clarify the effects of long-term high-fat diet on cognitive function, explore the effects and mechanisms of long-term high-fat diet on predation behavior in rats, and have guiding significance for people's health.

Many researchers use rodent predation behavior to explore the neurobiological mechanisms of cognitive function. It is often necessary to artificially train animals. Excessive training affects the behavioral autonomy of rats, thus affecting the results of predatory behavior. Reflects its true neurobiological nature. Therefore, the author used the laboratory predatory behavior observation model of Li et al. to eliminate the influence of human factors on the predation behavior of rats, and quantitatively determine the predation behavior of rats, and explore its neurobiological mechanism.

The open field experiment is a classic method for evaluating motor function and state anxiety in rodent experimental animals. We use this to determine that animal predation behavior disorder is not caused by changes in exercise capacity, inquiry behavior, animal mood, etc. The experimental results of the control group and the high-fat group showed that there was no significant difference between the two groups, suggesting that the high-fat diet had no effect on motor function in rats.

This study found that the predation of non-competitive predation behavior and competitive predation behavior in the high-fat group was significantly lower than that in the control group, suggesting that the rats in the high-fat group were reluctant to make efforts to obtain more food, indicating high. The fat diet does affect the predation behavior of rats.

Multiple brain regions of the central nervous system and a variety of chemicals are involved in regulating predatory behavior in rodents. The anterior cingulate cortex is located in the medial part of the prefrontal cortex and participates in the regulation of complex behaviors such as cognition, learning, and social assessment. Primate individuals with damaged anterior cingulate cortex do not correctly integrate risk and reward in enhanced mediated selection tasks. Rodent lesions in the anterior cingulate cortex of rodents indicate that rats with impaired anterior cingulate cortex give up the behavior of “get more and get more” and are more willing to spend less effort to get less income. Li Mingbo and other studies have shown that the predation behavior of the rats with bilateral anterior cingulate cortex damage is significantly impaired, and the competitive predation is the most obvious. The results of this study showed that the predation of rats in the high-fat group was significantly reduced. It is speculated that the predatory behavior disorder in rats with high-fat diet may be related to the changes of anterior cingulate cortex function. The c-Fos protein acts as a third messenger involved in intracellular signal transduction, and its expression reflects the activity of neurons on certain stimuli and is often used as a marker of neuronal activation. In this study, the expression of c-Fos in the anterior cingulate cortex was significantly decreased during the predation of high-dose rats, suggesting that the mechanism of long-term high-fat diet affecting predatory behavior in rats may be related to the decline of neuronal function in the anterior cingulate cortex; The mechanism still needs further discussion.