Effects of age and gender on recognizing ability and open field behavior of new objects in mice
YT(Xi'an) Biochem Co., Ltd. , https://www.ytnutra.com
Effects of age and gender on recognizing ability and open field behavior of new objects in mice
Summary
Objective [To study the effects of age and gender on the novel-object recognition ability and market activity of mice, and to understand the relationship between the two types of behavior. Methods [Experiment was completed in 2004-05 at the Behavioral Laboratory of Anhui Institute of Geriatrics. The 47 Kunming mice enrolled were divided into two groups: the young group (seven months and eight months old, 12 males, 11 females) and the elderly group (17, 8 months old, 12 males, 12 females). The non-spatial short-term memory ability of the mice was detected by the new object recognition task. The exploratory activity (locomotor) behavior of the mice was detected by the open field task, and the relationship between the tasks was judged by principal factor analysis. Results [In the new object recognition memory task, the new object priority index of the old female rats was (55.6±4.9)%, which was significantly lower than that of the young female rats (66.6±3.7)%. In the open field task, the exploration activity of the old female rats was reduced, and the time spent by the male and female aged rats around the open field [(294.4±2.0), (289.3±4.3) s] was compared with the corresponding young mice [( 278.2 ± 4.9), (267.2 ± 6.3) s] are more. The new object recognition score is positively correlated with the surrounding time in the market task, and negatively correlated with the cross-lattice number.
Conclusion [Female Kunming mice have the ability to recognize new objects (short-term memory) and are affected by the behavior of the open field.
Key words: aging; mice; learning; memory; behavior
0 Preface
Age-related memory decline in mice, especially spatial memory loss, has been studied in depth, and hippocampus has been well documented in spatial learning and memory [1-8]. However, the impact of aging on non-spatial learning and memory ability is mainly concentrated on the electric shock escape task. The recognition of new objects is a hippocampal-dependent task, but whether aging affects the memory has not been proven. Kunming mice are the most commonly used mice in Chinese laboratories, but their aging-related behavioral changes are rarely explored. In the current study, the authors used the new object recognition procedure to observe whether Kunming mice had age- or gender-related new object recognition (novel-object recognition). If so, whether it is affected by the open-field exploration behavior (locomotor) of a similar experimental environment.
1 Materials and methods
Design: Using experimental animals as the research object, randomized controlled and blinded trials using two-way analysis of variance.
Unit: Institute of Neurology and Geriatrics at a university hospital, Life Science Academy at a university.
MATERIALS: The experiment was performed at the Behavioral Laboratory of the Institute of Geriatrics, Anhui Province in 2004-05. The experimental mice were purchased from the Anhui Medical Laboratory Animal Center (皖医动92002). A total of 47 Kunming mice were divided into two groups according to age, 23 young rats of 7-8 months old (12 males and 11 females) and 24 old aged rats of 17 and 8 months old (12 females and 12 females). Mice were pre-selected prior to the start of the experiment, and mice with dyskinesia, hair loss, visible masses on the body surface, and large belly were excluded. Five weeks before the start of the experiment, the mice were housed in a plastic rat cage (size 25.5 cm × 15.0 cm × 14.0 cm) in the laboratory, and sawdust was placed in the cage. Among them, female rats are raised (four or five per cage), male, and single-raised. At least one mouse per day (tails, strokes). Animals were free to eat and enter water throughout the experiment unless specifically mentioned. The light-dark cycle of the feeding environment is 12h (lights are turned on at 7:00, lights are turned off at 19:00), room temperature is (21 ± 1) °C, and humidity is (55 ± 5)%. The experimenter did not know the grouping of mice. The experiment was completed in a bright cycle. One hour before the start of each experiment, the mice were moved from the breeding room to the testing room to maintain the adaptation of the mice to the environment.
Design, implementation, and evaluator: All authors of this article. The experiment was conducted using a 3-blind method in which the mice's groupers, experiments, evaluators, and data statisticians were independently completed by three individuals, and the evaluators were professionally trained laboratory workers.
METHODS: Instruments and behavioral procedures were partially modified according to previously published methods. The authors designed a market behavioral procedure to measure spontaneous activity and anxiety in mice [9], a new object recognition behavior procedure to determine Non-spatial short-term memory in mice [10].
Market: The instrument is an open black wooden box (the internal size is 81cm × 81cm), and the wall is 28cm high. The bottom of the box is painted in three vertical and horizontal lines (width 3 mm) to form 16 equal squares (20 cm x 20 cm each). A color box (8 cm x 5 cm x 3 cm) was placed in the center of the field to stimulate the curiosity of the mice. The lighting comes from a 40w incandescent lamp that is 2.8m above the center of the venue. Each mouse received one test, during which the mice placed one of the four corner squares facing the wall and allowed them to freely explore the environment for 5 min. After the end of each mouse test, rinse the water with water and dry.
New object recognition: The device is an open wooden box painted in black (50cm × 50cm × 25cm). An incandescent lamp provides illumination at a bottom level of approximately 150 lx. During the test, the mice were placed in empty boxes and allowed to explore for 5 minutes freely to adapt to the environment. The mice were then transferred to a breeding cage. After 5 minutes, the mice returned to the box and entered the training period. At this time, there are two identical objects (color carton, size 6.0 cm × 6.0 cm × 3.5 cm) which are linearly arranged along a wall 8 cm from the wall of the box. Mice were placed in the box from one of the other two corners and returned to the cage after 5 minutes of free exploration. After a 5 min interval, the animals were reintroduced into the experimental box for free exploration during the experimental period. At this time, one of the two used color cartons during the training period was replaced by a purple box (7.0 cm × 7.0 cm × 5.5 cm) and placed in the original position, while another familiar color carton was moved to the new one. The opposite corners of the object prevent the bias of the mouse space. If the mouse points its object or touches the nose within 1 cm with its nose, it is considered an exploration behavior.
MAIN OUTCOME MEASURES: The open field task was to record the total number of squares spanned by the mouse during the experimental period and the surrounding time (the total time spent on the surrounding 12 squares). In the new object recognition task, the time of exploring the familiar object (TF) and exploring the new object (TN) during the experimental period is recorded separately. The priority index (PLN) for new objects during the experimental period is defined as TN/(TF+TN)*100%. Similarly, the total time of exploration of object 1 (T01) and object 2 (T02), and the priority index of object 1 (PI01) and object 2 (PI02) are also recorded separately during the training period. The total exploration time for the training period (TTTR) and the experimental period (TTTE) also entered the statistical analysis.
Statistical analysis: All results are expressed as mean ± standard error. The statistical method used was a two-way analysis of variance, with age and gender being independent variables. In order to avoid confusion caused by different gender patterns in the decline of age-related scores in the two-way analysis of variance, the one-sex analysis of variance (ie, one-way analysis of variance for each gender) was used to show the age-primary effect [2]. Similarly, the “single age†analysis of variance, one-way analysis of variance for each age, was used to show gender main effects.
In order to explore the interrelationship between tasks and tasks, the authors used principal factor analysis techniques [2, 11] similar to previous studies [2, 11], the initial factor model was transformed by the Varimax method, and the Eigen value was greater than 1. Reserved. P < 0.05 was considered significant. Statistical analysis was performed by the first author by SPSS for window 11.5 software.
2 results
2.1 Analysis of the number of experimental animals Due to the short cycle, the experiment almost did not cause stress, hunger effects and physical exertion on the animals. All 47 mice were lost and died in the middle, and all the animals' data entered the results analysis.
2.2 Results of open field experiment The mean peripheral time of old female and male rats was significantly longer than that of the young rats [F(1,21)=9.96, P<0.01; F(1,21)=8.42, P<0.01]. These differences were mainly due to age effect [F(1,45)=17.44, P<0.01], while the effect of gender was smaller [F(1,45)=2.99, P=0.091]. The interaction between age and gender is minimal [F(1,45)<1].
Age and gender did not affect the cross-number of mice [F(1,45)=2021,1.09; Ps>0.05], and the interaction between age and gender had no effect on the cross-grid [F(1,45)< 1]. However, the “single sex†analysis of variance showed that the locomotor activity of older females was significantly lower than that of young females [F(1,21)=4.87, P<0.05], but the “single age†analysis of variance showed There was no significant difference in the number of crossovers between male and female mice [F(1,21)=2.08, P>0.05].
2.3 New object recognition experiment results Two-way analysis of variance showed that TTTR and TTTE were not affected by age [F(1,45)=2.21, 1.09; Ps>0.05] and gender [F(1,45)=0.24, 2.78; Ps> The effect of 0.05], but was strongly influenced by age-sex interaction [F(1,43)=18.39,8.02; Ps<0.001,0.01]. Further “single-sex†analysis of variance and “single-age†analysis of variance showed that TTTR and TTTE were significantly shorter in older females than in young females [F(1,21)=14.08, 9.34; P=0.001, <0.01] and elderly Male rats [F(1,22)=14.03,6.84; P=0.001,<0.05], but young female rats had longer TTTR than males of the same age [F(1,21)=6.08, 9.34; P<0.05 ] and similar TTTE [F (1, 21) = 1.31, 6.84; P > 0.05]. However, age, gender, and age-to-sex interaction did not significantly affect PI01 and PI02 [F(1,45/43)<1].
For the priority index, repeated measures analysis of variance showed that PIn was significantly higher than PI01 or PI02 [F(1,46)=18.77, 14.81; Ps<0.001] when all animals were analyzed together. Tip In the open field, Kunming mice have the ability to remember familiar objects and get used to preferentially exploring new objects. The age affecting this effect [F(1,45)=3.97, P=0.052] instead of gender [F(1,45)<1] and age-sex interaction [F(1,43)<1]. Two-way analysis of variance showed that age [F(1,45)=6.41, P<0.05] significantly affected PIn, while gender [F(1,45)<1] and gender-age interaction had no effect [F(1) , 43) <1]. The “single sex†analysis of variance showed that the PIn of the older female rats was significantly smaller than that of the young females [F(1, 21) = 4.47, P < 0.05].
2.4 The Kaiser-Meyer-Olkin measure of sampling adequacy is 0.665 (>0.05), and P<0.001 in the Bartlett spherical hypothesis, indicating that the selected index in this study is suitable for the main factor. analysis. As a result, three significant factors were obtained. The component load of the different variables rotated on each factor. Within the task, the five indicators of the new object recognition task belong to three different factors, that is, the total exploration time of the training period and the experimental period is rotated by the factor 2, and the priority index of the object 1 and the object 2 is rotated by the factor during the training period. 1 But the direction is opposite, and the priority index rotation of the new object during the experimental period is equal to the factor 3; the two indicators of the open field task are rotated by the factor 3, and the direction is also opposite. Between tasks, the new object priority index and the surrounding time are rotated in the same direction as the factor 3, and the cross-grid number is also rotated in the same direction as the factor 3.
3 Discussion
3.1 Mobile exploration of aged mice The active open field task is one of the tasks frequently applied to the behavioral study of aging mice [1, 2, 12]. The results of this paper show that the mobile exploration activity represented by the cross-grid number is lower in older females than in young females. Using the open field or beam active system, previous studies reported reduced activity in mobile exploration in many other strains of aging mice [2, 4, 12]. The exploratory behavior that represents the activity of mobile exploration is mainly along the side walls of the site, although there is a novel object (a color carton used to stimulate the curiosity of the mouse) located in the center of the field. The peripheral time of the elderly Kunming mice was significantly longer than that of the young rats, suggesting that the anxiety of elderly Kunming mice is higher than that of young mice. This is consistent with observations in elderly rats and middle-to-old male and female NMRI mice in the elevated plus maze [12,13]. The authors believe that the decrease in mobile exploration activity in elderly Kunming mice is likely to be due to an increase in anxiety, rather than as other researchers have specifically mentioned in the study of elderly OF1 and NMRI females due to reduced exploration activity and curiosity. Heart loss and decreased physical fitness, because although the anxiety of both male and female Kunming mice increased, only the mobile exploration activity of older females decreased [11,12]. The elderly Kunming mice in this paper showed similar anxiety results in the open field. The results of the elderly C57BL/6NIA mice in the elevated plus maze were slightly different, that is, the 17-month-old C57BL/6NIA female mice. Middle-aged mice are more anxious than males of the same age and young (5-month-old) females. However, the anxiety of older rats (25 months old) is similar to that of young rats [2]. Because the market behavior of mice has some similarities with the food storage behavior of mice, the age-related changes in the behavior of Kunming mice may be affected by changes in age-related food storage behavior [14].
3.2 Changes in non-spatial cognitive ability of elderly Kunming mice In this study, the authors used the new object recognition task to determine whether the non-spatial learning and memory ability of Kunming mice changed with aging. This task is one of the hippocampal-dependent tasks [10] and is rarely used for the evaluation of cognitive ability in aging mice. Older females have smaller PIN than young females, suggesting that some aspects of non-spatial short-term memory in older male and female Kunming mice are impaired. Recently, the effects of different exploration times on PIn have been studied. In order to obtain PIn, it has been recommended that the total exploration time be accumulated to the same level [15]. In older females, although the possibility of a short exploration time on the cognitive impairment of older females cannot be completely ruled out, the poor performance of older females in the task of recognizing new objects is likely to originate from their own. Cognitive impairment. The reason is that although the total time of exploration of the old female rats during the training period and the experimental period is indeed shorter than that of the young female rats, the two groups of mice spent 50% of the time on the object 1 and the object 2 during the training period. During the experimental period, although young females spent 67% of the time on new objects (Pin = 0.667), older females spent only 56% of the time on new objects (Pin = 0.556). This suggests that older females may not be able to distinguish between familiar and new objects due to cognitive impairment, resulting in too little time spent on new objects.
3.3 Gender Differences in Cognitive Tasks Gender differences in spatial learning and memory abilities have been well documented in various species [2, 16, 17]. In the eight-arm radial maze task, young females performed better than males [17]. For normal aging mice, there is little data directly showing gender differences in cognitive decline. Frick et al [2] have found that 17-month-old female C57BL/6NIA mice show more spatial reference memory impairment in Morris, but there is no gender difference in 25-month-old mice. The results of this paper suggest that some non-spatial cognitive abilities, such as the age-related decline in new object recognition ability, are earlier in females than in males. It is worth mentioning that, as discussed above, the gender and age-related non-spatial learning and memory impairments shown in this experiment may be due to different feeding environments between female (group) males (monoculture) mice. . However, the feeding effect may not be the main confounding factor. The reason is: 1 The damage of age-related learning and memory ability occurs only in the group of old females rather than the single-raised old males. 2 In the task of recognizing new objects, the results of older male rats are better than those of older females. 3 single-raised young males performed better than group young females. Of course, the final conclusion still needs to be obtained by single or group male and female rats.
3.4 Inter-task and inter-task indicator interrelationships The main factor analysis has been used to study the nature of older and younger mice to distinguish between different learning and memory task indicators, but it is not used to distinguish the nature of learning and memory tasks and sensory motor task indicators. [3]. The results of this paper show that the indicators in the new object recognition task may be different aspects of cognitive function, while the market task determines the opposite direction of sensory motor function. Some previous studies have attempted to determine the interrelationships between tasks within different behavioral task groups, but the conclusions reached are mutually contradictory [12,18]. In one task group, only the higher æ—· field motility of 17- to 22-month-old NMRI mice was found to be inversely related to their performance in the Morris water maze [12]. In another study, the interrelationship between many tasks was discovered [3]. In the task group of this paper, the market is measuring the components of mobile exploration activities. Obviously, the new object recognition task is also explored by the obvious movement.
Conclusion: The results of this paper show that the recognition of new objects is positively correlated with the number of grids in the field and negatively correlated with the surrounding time. This suggests that the stronger the mobile exploration activity of Kunming mice, the stronger the recognition ability of new objects. In contrast, the greater the anxiety of Kunming mice, the worse their ability to recognize new objects.