Abstract: Objective To study the effects of limiting thermal energy intake (limited food) on behavior and cognition in growing rats. Â
Methods Weaned SD rats (male and female) were randomly divided into 3 groups: free feeding group, 20% restriction group (20% CR), and 35% restriction group (35 % CR). At 3 and 6 months, the behavior and cognitive ability of the rats were tested by open field test and Y2 maze method. After 6 months, the rats were sacrificed. The content of dopamine (DA), 52 hydroxytryptamine (52HT) and norepinephrine (NE) in brain stem and telencephalon were determined by fluorescence spectrophotometry. The cortex, hippocampus and cerebellar nitric oxide were determined. Enzyme (NOS) activity. Â
Results The weight of female and male rats in the restricted diet group was significantly lower than that in the control group. Male rats were restricted for 3 months. The number of moving grids in the 20% CR group was significantly lower than that in the control group and 35% CR group. The limit of 6 months was limited, and the number of moving grids in the 35% CR group was significantly higher than that in the control group and 20% CR group. The restriction of food had no significant effect on the test of females in the open field. Dieting for 6 months had no significant effect on learning and memory performance in rats. There was no significant difference between the three groups of brain DA, 52HT, NE content and NOS activity after 6 months of restriction. Â
Conclusion The effect of food restriction on the exploration activities of rats in the growing period is gender-differentiated. The food restriction delays the decrease of short-term activity of male rats, but has no effect on female rats. Dieting for 6 months had no significant effect on learning and memory performance in rats. Â
Keywords: food restriction, cognition, behavior, rat Â
Numerous studies have shown that food restriction can prolong the lifespan of many animals [1], delay or reduce the occurrence of degenerative diseases including cancer, diabetes and neurological disorders [2,3]. Food restriction can also slow the decline of learning and memory and motor function in rodents, and improve the behavior of experimental animal models with neurodegenerative diseases [4, 5]. However, most of these studies focus on older animals, focusing on the beneficial effects of food restriction, thus ignoring the adverse effects of food restriction on growth and development, especially on their behavior and cognitive function. Therefore, in this study, weaned SD rats were used as experimental subjects to observe the effects of 3 and 6 months of restriction on the cognition and behavior of female and male rats. Â
1 Materials and methods Â
1.1 Experimental animals Â
Clean-grade weaning SD rats, male and female, were provided by Shanghai Xipuer 2 Bikai Experimental Animal Co., Ltd. After 3 days of adaptation, they were randomly divided into 3 groups: free feeding group (control); food restriction group 20% (20% CR), energy intake was 80% of the control group; food restriction group 35% (35 % CR), energy intake Into the control group of 65%. There were 15 males and 16 females in each group. The artificially prepared feed (Table 1) was fed in a single cage and freely taken. The feed was started every day at 8:00 am, and the actual intake of the feed in the free feeding group was weighed to calculate the amount of feed for the animals in the restricted diet group the next day. The room temperature is controlled at 18-22 ° C, the humidity is 45% to 50%, and the illumination time is (12 ± 1) h. Body weight changes were recorded weekly for 6 months during the experimental period. The feed was prepared from casein, starch, soybean oil, mixed vitamins and mixed inorganic salts. The intake of other nutrients was consistent among the three groups except for the intake of carbohydrates. Â
1.2 Observation indicators and methods Â
1.2.1 Field test The rats were placed in the center of the open field with a diameter of 90 cm and 16 equal parts, and the number of moving grids and the number of standings were observed within 3 minutes. Â
1.2.2 Learning and memory behavior detection using the Y 2 maze method. Pre-selected rats with more consistent response to electric shock, tested learning ability, and tested memory ability after 48 hours. After the rat was shocked, the escape from the starting area directly to the safe area was “correct responseâ€. The correct response was achieved 9 times in 10 consecutive times, and the number of electric shocks required before reaching the standard was recorded as the learning and memory score. Â
1.2.3 Determination of dopamine (DA), 52 hydroxytryptamine (52HT), norepinephrine (NE) content Â
At the end of the experiment, the animals were sacrificed and the brainstem and telencephalon of the brain tissue were separated. The content of DA, 52HT and NE in each fraction was determined by fluorescence spectrophotometry. The excitation P emission lengths of DA, 52HT, and NE are: 329P 372, 359P 449, and 385P 484, respectively. Â
1.2.4 Determination of brain nitric oxide synthase (NOS) activity At the end of the experiment, the animals were sacrificed and the cortex, hippocampus and cerebellum of the brain tissue were isolated. The NOS assay was performed. Protein quantification was performed using Lowry's method according to the kit instructions [6]. Â
1.3 Statistical processing Â
The experimental data were expressed as x±s, and the variance analysis of the results was performed using SPSS1210 software. The significance limit value was P < 0105. Â
2 results Â
2.1 Growth and development Â
Restricted animals showed irritability within 1 to 2 weeks after the start of the restriction, and there was more activity. No abnormalities were observed subsequently, and no abnormalities were observed in the animal anatomy. At 6 months, the weight of female and male rats in the restricted diet group was significantly lower than that in the control group, and the body weight of the 35% CR group was significantly lower than that in the 20% CR group. Â
2.2 Market test Â
Male rats were restricted for 3 months, and the number of 20% CR moving grids was significantly lower than that of the control group and the 35% CR group. After 6 months of restriction, the number of moving grids in the 35% CR group was significantly higher than that in the control group and 20% CR group. The limit food has no significant effect on the number of standing males and the number of moving lattices and standing times of females. Â
2.3 The effect of food restriction on learning and memory ability Â
Female rats were limited to 3 months, and the learning scores of 20% CR group were significantly higher than those of the control group. There was no significant difference in memory scores between the three groups. Female rats were limited to 6 months and male rats were restricted for 3 or 6 months. The results have no significant effect. Â
2.4 The effect of food restriction on the content of DA, 52HT and NE in the brain showed that there was no significant difference between the three groups of brainstem and cerebral brain DA, 52HT and NE. Â
2.5 The effect of food restriction on NOS content in brain showed that 6-month limit food had no significant effect on NOS activity in rat cortex, hippocampus and cerebellum. Â
3 Discussion Â
According to previous studies [7,8], the limit foods designed in this experiment were the control group, 20% CR and 35% CR group. The results showed that limiting energy intake inhibited the weight gain of female and male rats, and the more severe the restriction, the greater the degree of inhibition. Mine tests are often used to reflect the animal's ability to explore and short-term activity. In the 6-month limit, the short-term activity of the 35% CR group was significantly higher than that of the free food intake group. The results of this study showed the same trend as the Yu study [9], that is, the food restriction energy was delayed with the decrease in activity. This may have a role in reducing fat accumulation, enhancing cardiovascular function, reducing the incidence of geriatric diseases, and prolonging life. The restriction of food has no effect on the short-term activity of female rats, indicating that the effect of food restriction on the exploration behavior and short-term activity has gender differences. The reason may be related to the different adaptability of male and female to food restriction, which needs further study. Limiting food for 3 months can significantly improve the academic performance of 20% of CR females. Food restriction has no effect on the learning performance of male rats. Previous studies have shown that limiting the diet for 3 months and 6 months can improve the learning ability of male mice, but has no effect on their memory ability and learning and memory ability in female mice [10]. Thus, the effect of food restriction on animal performance may be related to age and species. Dietary food has no obvious effect on the content of DA, NE and 52HT in brainstem and telencephalon, which is basically consistent with the results of learning and memory. However, whether food restriction affects other neurotransmitters related to learning and memory, such as acetylcholine, and the discussion of deeper mechanisms are the next step. Limitation of food for six months had no significant effect on NOS activity in the brain, suggesting that food restriction had no effect on NO biological function in the brain, which was basically consistent with the results of learning and memory tests. In conclusion, the results of the study showed that the effect of food restriction on the behavioral behavior of rats was gender-differentiated, and the restriction of food was delayed in the reduction of short-term activity of male rats, which had no effect on female rats. Dieting for 6 months had no significant effect on learning and memory performance in rats. Â
Methods Weaned SD rats (male and female) were randomly divided into 3 groups: free feeding group, 20% restriction group (20% CR), and 35% restriction group (35 % CR). At 3 and 6 months, the behavior and cognitive ability of the rats were tested by open field test and Y2 maze method. After 6 months, the rats were sacrificed. The content of dopamine (DA), 52 hydroxytryptamine (52HT) and norepinephrine (NE) in brain stem and telencephalon were determined by fluorescence spectrophotometry. The cortex, hippocampus and cerebellar nitric oxide were determined. Enzyme (NOS) activity. Â
Results The weight of female and male rats in the restricted diet group was significantly lower than that in the control group. Male rats were restricted for 3 months. The number of moving grids in the 20% CR group was significantly lower than that in the control group and 35% CR group. The limit of 6 months was limited, and the number of moving grids in the 35% CR group was significantly higher than that in the control group and 20% CR group. The restriction of food had no significant effect on the test of females in the open field. Dieting for 6 months had no significant effect on learning and memory performance in rats. There was no significant difference between the three groups of brain DA, 52HT, NE content and NOS activity after 6 months of restriction. Â
Conclusion The effect of food restriction on the exploration activities of rats in the growing period is gender-differentiated. The food restriction delays the decrease of short-term activity of male rats, but has no effect on female rats. Dieting for 6 months had no significant effect on learning and memory performance in rats. Â
Keywords: food restriction, cognition, behavior, rat Â
Numerous studies have shown that food restriction can prolong the lifespan of many animals [1], delay or reduce the occurrence of degenerative diseases including cancer, diabetes and neurological disorders [2,3]. Food restriction can also slow the decline of learning and memory and motor function in rodents, and improve the behavior of experimental animal models with neurodegenerative diseases [4, 5]. However, most of these studies focus on older animals, focusing on the beneficial effects of food restriction, thus ignoring the adverse effects of food restriction on growth and development, especially on their behavior and cognitive function. Therefore, in this study, weaned SD rats were used as experimental subjects to observe the effects of 3 and 6 months of restriction on the cognition and behavior of female and male rats. Â
1 Materials and methods Â
1.1 Experimental animals Â
Clean-grade weaning SD rats, male and female, were provided by Shanghai Xipuer 2 Bikai Experimental Animal Co., Ltd. After 3 days of adaptation, they were randomly divided into 3 groups: free feeding group (control); food restriction group 20% (20% CR), energy intake was 80% of the control group; food restriction group 35% (35 % CR), energy intake Into the control group of 65%. There were 15 males and 16 females in each group. The artificially prepared feed (Table 1) was fed in a single cage and freely taken. The feed was started every day at 8:00 am, and the actual intake of the feed in the free feeding group was weighed to calculate the amount of feed for the animals in the restricted diet group the next day. The room temperature is controlled at 18-22 ° C, the humidity is 45% to 50%, and the illumination time is (12 ± 1) h. Body weight changes were recorded weekly for 6 months during the experimental period. The feed was prepared from casein, starch, soybean oil, mixed vitamins and mixed inorganic salts. The intake of other nutrients was consistent among the three groups except for the intake of carbohydrates. Â
1.2 Observation indicators and methods Â
1.2.1 Field test The rats were placed in the center of the open field with a diameter of 90 cm and 16 equal parts, and the number of moving grids and the number of standings were observed within 3 minutes. Â
1.2.2 Learning and memory behavior detection using the Y 2 maze method. Pre-selected rats with more consistent response to electric shock, tested learning ability, and tested memory ability after 48 hours. After the rat was shocked, the escape from the starting area directly to the safe area was “correct responseâ€. The correct response was achieved 9 times in 10 consecutive times, and the number of electric shocks required before reaching the standard was recorded as the learning and memory score. Â
1.2.3 Determination of dopamine (DA), 52 hydroxytryptamine (52HT), norepinephrine (NE) content Â
At the end of the experiment, the animals were sacrificed and the brainstem and telencephalon of the brain tissue were separated. The content of DA, 52HT and NE in each fraction was determined by fluorescence spectrophotometry. The excitation P emission lengths of DA, 52HT, and NE are: 329P 372, 359P 449, and 385P 484, respectively. Â
1.2.4 Determination of brain nitric oxide synthase (NOS) activity At the end of the experiment, the animals were sacrificed and the cortex, hippocampus and cerebellum of the brain tissue were isolated. The NOS assay was performed. Protein quantification was performed using Lowry's method according to the kit instructions [6]. Â
1.3 Statistical processing Â
The experimental data were expressed as x±s, and the variance analysis of the results was performed using SPSS1210 software. The significance limit value was P < 0105. Â
2 results Â
2.1 Growth and development Â
Restricted animals showed irritability within 1 to 2 weeks after the start of the restriction, and there was more activity. No abnormalities were observed subsequently, and no abnormalities were observed in the animal anatomy. At 6 months, the weight of female and male rats in the restricted diet group was significantly lower than that in the control group, and the body weight of the 35% CR group was significantly lower than that in the 20% CR group. Â
2.2 Market test Â
Male rats were restricted for 3 months, and the number of 20% CR moving grids was significantly lower than that of the control group and the 35% CR group. After 6 months of restriction, the number of moving grids in the 35% CR group was significantly higher than that in the control group and 20% CR group. The limit food has no significant effect on the number of standing males and the number of moving lattices and standing times of females. Â
2.3 The effect of food restriction on learning and memory ability Â
Female rats were limited to 3 months, and the learning scores of 20% CR group were significantly higher than those of the control group. There was no significant difference in memory scores between the three groups. Female rats were limited to 6 months and male rats were restricted for 3 or 6 months. The results have no significant effect. Â
2.4 The effect of food restriction on the content of DA, 52HT and NE in the brain showed that there was no significant difference between the three groups of brainstem and cerebral brain DA, 52HT and NE. Â
2.5 The effect of food restriction on NOS content in brain showed that 6-month limit food had no significant effect on NOS activity in rat cortex, hippocampus and cerebellum. Â
3 Discussion Â
According to previous studies [7,8], the limit foods designed in this experiment were the control group, 20% CR and 35% CR group. The results showed that limiting energy intake inhibited the weight gain of female and male rats, and the more severe the restriction, the greater the degree of inhibition. Mine tests are often used to reflect the animal's ability to explore and short-term activity. In the 6-month limit, the short-term activity of the 35% CR group was significantly higher than that of the free food intake group. The results of this study showed the same trend as the Yu study [9], that is, the food restriction energy was delayed with the decrease in activity. This may have a role in reducing fat accumulation, enhancing cardiovascular function, reducing the incidence of geriatric diseases, and prolonging life. The restriction of food has no effect on the short-term activity of female rats, indicating that the effect of food restriction on the exploration behavior and short-term activity has gender differences. The reason may be related to the different adaptability of male and female to food restriction, which needs further study. Limiting food for 3 months can significantly improve the academic performance of 20% of CR females. Food restriction has no effect on the learning performance of male rats. Previous studies have shown that limiting the diet for 3 months and 6 months can improve the learning ability of male mice, but has no effect on their memory ability and learning and memory ability in female mice [10]. Thus, the effect of food restriction on animal performance may be related to age and species. Dietary food has no obvious effect on the content of DA, NE and 52HT in brainstem and telencephalon, which is basically consistent with the results of learning and memory. However, whether food restriction affects other neurotransmitters related to learning and memory, such as acetylcholine, and the discussion of deeper mechanisms are the next step. Limitation of food for six months had no significant effect on NOS activity in the brain, suggesting that food restriction had no effect on NO biological function in the brain, which was basically consistent with the results of learning and memory tests. In conclusion, the results of the study showed that the effect of food restriction on the behavioral behavior of rats was gender-differentiated, and the restriction of food was delayed in the reduction of short-term activity of male rats, which had no effect on female rats. Dieting for 6 months had no significant effect on learning and memory performance in rats. Â
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