CN113940286A - Method for training and improving space learning capacity of mouse - Google Patents
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- 238000012549 training Methods 0.000 title claims abstract description 66
- 230000013016 learning Effects 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000011765 DBA/2 mouse Methods 0.000 claims abstract description 62
- 238000011740 C57BL/6 mouse Methods 0.000 claims abstract description 36
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 32
- 241000699670 Mus sp. Species 0.000 claims abstract description 24
- 230000006872 improvement Effects 0.000 claims abstract description 19
- 238000002474 experimental method Methods 0.000 claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 10
- 238000011156 evaluation Methods 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 101100294532 Mus musculus Nr1d1 gene Proteins 0.000 claims abstract description 5
- 238000010832 independent-sample T-test Methods 0.000 claims abstract description 5
- 230000014509 gene expression Effects 0.000 claims description 29
- 230000031836 visual learning Effects 0.000 claims description 27
- 101150081376 NR1D1 gene Proteins 0.000 claims description 15
- 210000001320 hippocampus Anatomy 0.000 claims description 9
- 238000012347 Morris Water Maze Methods 0.000 abstract description 19
- 239000003814 drug Substances 0.000 abstract description 4
- 239000003550 marker Substances 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 3
- 230000000971 hippocampal effect Effects 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 7
- 230000015654 memory Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000010171 animal model Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000006886 spatial memory Effects 0.000 description 3
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- 101100259948 Mus musculus Tbata gene Proteins 0.000 description 1
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- A01K15/00—Devices for taming animals, e.g. nose-rings or hobbles; Devices for overturning animals in general; Training or exercising equipment; Covering boxes
- A01K15/02—Training or exercising equipment, e.g. mazes or labyrinths for animals ; Electric shock devices ; Toys specially adapted for animals
- A01K15/027—Exercising equipment, e.g. tread mills, carousels
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention provides a method for training and improving the space learning capacity of a mouse, which is characterized in that the mouse is trained by utilizing a water maze, the training period is 30 days and 2 times per day, repeated measurement variance analysis is adopted from the latency period before the DBA/2 mouse experiment to the latency period after the experiment, and the analysis and the latency period of the C57BL/6 mouse are analyzed by adopting independent sample t test, so that the improvement effect evaluation of the space learning capacity of the DBA/2 mouse is realized. The invention also provides application of the mouse Nr1d1 gene in improving the space learning ability of mice. The invention doubly measures the improvement of the Morris water maze training on the DBA/2 mouse space learning ability, and provides a molecular biology evaluation marker which can be used as a training or medicine for improving the mouse space learning ability effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for training and improving the space learning capacity of a mouse.
Background
The spatial learning memory is a behavior pattern for detecting the abilities of spatial orientation, reaction time, visual perception, structural application and the like so as to evaluate the cognitive level of the spatial learning memory, and is a type of learning memory about scenes and time. The Morris Water Maze (MWM) is a device designed by the psychologist Morris in the early 80 s of the 20 th century for researching the spatial learning and memory of rodents, the MWM is used as a detection means for the spatial learning and memory capacity of animals in the past research, and the influence of repeated MWM training on the spatial learning and memory capacity of the animals is rarely concerned. In fact, the influence of repeated MWM training on the spatial learning and memory ability has important practical significance. In real life, space learning and memory capacity often needs to be continuously strengthened and repeated to finally stabilize, and the space learning and memory capacity becomes fixed memory and provides help and guidance for daily life of people.
DBA/2 and C57BL/6 mice are two inbred line varieties which are most widely applied in the field of neuroscience, and a large number of researches prove that the DBA/2 mice have the performance which is obviously worse than that of the C57BL/6 mice in the aspects of space learning and memory depending on hippocampal tissues, such as the Morris water maze and the like. The behavior and genetic differences of the two identified mice are very important for neuroscience research, and the different expression patterns of certain genes in hippocampal tissues are also shown to be possibly related to the spatial learning and memory ability of the animals.
The repeated exposure to the spatial learning stimulus is found to improve the subsequent spatial learning ability of the animal, but the further research of discussing the expression of genes related to the improvement of the spatial learning ability of the mouse by repeated training in the hippocampal tissue from the level of molecular biology is lacked, the spatial learning ability of the animal is generally evaluated by comparing the results of the animal training before and after, and the animal with stronger spatial learning ability is not used as a measuring pole.
Disclosure of Invention
In order to overcome the defects in the prior art, the method for training and improving the space learning capacity of the mouse is provided. The method evaluates the space learning ability of DBA/2 and C57BL/6 mice by performing a water maze directional navigation experiment with a period of 6 days and measuring the incubation period of the upper stage; carrying out continuous 30-day oriented sailing training on DBA/2 and C57BL/6 in a water maze, and measuring the improvement of space learning capacity of DBA/2 mice according to the shortening of the latency period in the training process of the DBA/2 mice and the comparison of the latency period after training and the latency period of C57 BL/6; the high-throughput sequencing technology is utilized to carry out transcriptome sequencing technology on hippocampal tissues of untrained DBA/2 mice, untrained C57BL/6 mice, trained DBA/2 mice and trained C57BL/6, important genetic information related to the improvement of DBA/2 mouse space learning capacity by repeated Morris water maze training is screened, the important genetic information can be used as a molecular biology evaluation marker for training the improvement of mouse space learning capacity effect, and an important reference can be provided for the evaluation of the medicine improvement of mouse space learning capacity effect
In order to solve the technical problems, the invention provides the following technical scheme that the method for training and improving the space learning capacity of the mouse utilizes the water maze to train the mouse, and the training period is more than 6-30 days and 1-2 times per day.
Preferably, the training period is 6-30 days.
Preferably, the training period is more than 30 days.
Preferably, the mouse is a DBA/2 mouse or a C57BL/6 mouse.
Preferably, the mice are 4-8 weeks old.
Preferably, repeated measurement analysis of variance is adopted from the latency period before the DBA/2 mouse experiment to the latency period after the experiment, and the latency period of the C57BL/64-8 mouse and the latency period are analyzed by adopting an independent sample t test, so that the improvement effect evaluation on the space learning capacity of the DBA/2 mouse is realized.
Preferably, the expression level of the Nr1d1 gene in the mouse hippocampus before and after the DBA/2 mouse experiment is counted and compared with the expression level of the C57BL/6 mouse Nr1d1 gene in the mouse hippocampus, so that the improvement effect evaluation of the space learning capacity of the DBA/2 mouse is realized.
The invention also aims to provide application of the mouse Nr1d1 gene in improving the space learning ability of mice.
The invention has the beneficial effects that:
according to the method, a C57BL/6 mouse with space learning capacity obviously higher than that of a DBA/2 mouse is selected as a measuring marker post by utilizing Morris water maze directional navigation detection, the improvement of the space learning capacity of the DBA/2 mouse is doubly measured by Morris water maze training through comparison of performances of the DBA/2 mouse at different stages and comparison of the results with the latency of the C57BL/6 mouse, then a transcriptome sequencing technology is carried out on hippocampal tissues of untrained DBA/2 mouse, trained DBA/2 mouse and trained C57BL/6 by utilizing a high-throughput sequencing technology, important genetic information related to the improvement of the space learning capacity of the DBA/2 mouse by repeated Morris water maze training is screened, and the important genetic information can be used as a molecular biology evaluation mark for training or medicine improvement of the space learning capacity effect of the mouse.
Drawings
Figure 1 is the difference in spatial learning capacity between DBA/2 and C57BL/6 mice (n = 10);
fig. 2 is the improvement of spatial learning capacity of DBA/2 mice by Morris water maze training (n = 10); wherein A is the incubation period results of mice 10 days before, 10 days in the middle and 10 days after training; b is that the latency of DBA/2 mice and C57BL/6 mice has no significant difference (P is more than 0.05) on day 30, the space learning ability of DBA/2 mice is significantly improved, and the level is equivalent to that of C57BL/6 mice;
fig. 3 shows the difference in expression of Nr1d1 gene in DBA/2 and C57BL/6 mouse hippocampal tissues (n = 3);
FIG. 4 shows the difference in expression of the Nr1d1 gene in hippocampus between untrained DBA/2 and trained DBA/2, C57BL/6 mice (n = 3).
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Example 1
The invention proves the relevance of the Nr1d1 gene and the water maze training for improving the space learning ability of mice through the following experiments.
1) Difference in spatial learning Capacity between DBA/2 and C57BL/6 mice
The experimental animals adopted are 20 DBA/2 and C57BL/6 mice each with 8 weeks of age, male, are bred for 7 days adaptively, then 10 DBA/2 mice and 10C 57BL/6 mice are selected to be subjected to Morris water maze positioning navigation test with 6 days of action, 1 time per day, the latency period (the time required for the first successful landing of the platform after water entry) of each mouse is recorded, the comparison of the latency periods of the DBA/2 mice and the C57BL/6 mice is analyzed by using an independent sample t test, and when p is less than 0.05, the difference is considered to be significant, namely, the difference has statistical significance.
Taking another 10 DBA/2 mice and 10C 57BL/6 mice, after adaptive feeding is finished, killing the C57BL/6 mice and the DBA/2 mice under an anesthesia state, separating hippocampal tissues, combining into 3 samples, extracting total RNA samples, constructing gene expression profiles of two groups of mouse hippocampal tissues by using a transcriptome sequencing technology, counting gene expression levels by using an FPKM method, and screening the differential expression genes of the two groups of mouse hippocampal tissues according to the following standard: the expression variation multiple is more than 1.2 times, and the corrected P value is less than 0.05. The results are shown in FIG. 3.
The results in FIG. 1 show that as the experiment progresses, the latency of the C57BL/6 mice is in overall downward trend, the DBA/2 mice are not in obvious trend, the latency difference of the two mice at days 1, 3 and 4 is not obvious (P > 0.05), but the latency of the DBA/2 mice is obviously longer than that of the C57BL/6 mice at days 2, 5 and 6 (P <0.05 and P < 0.001), which indicates that the space learning ability of the DBA/2 mice is obviously lower compared with that of the C57BL/6 mice.
2) Improvement of space learning capacity of DBA/2 mouse by Morris water maze training
The experimental animals adopted are 20 DBA/2 mice with the age of 4 weeks, 20C 57BL/6 mice and males, the experimental animals are bred for 7 days in an adaptive mode, the 20 DBA/2 mice are randomly divided into a D2 control group and a D2 training group, the 20C 57BL/6 mice are randomly divided into a B6 control group and a B6 training group, the two training groups are subjected to Morris water maze training for 30 days, the training time is 2 times per day, the incubation period of each mouse is recorded, and after the training is finished, the age of the mice is about 8 weeks. The mice in the D2 training group are compared before and after the incubation period in a repeated measurement ANOVA LSD method in pairs, the mice in the D2 training group are compared with the mice in the B6 training group in the incubation period in an independent sample t test, and when p is less than 0.05, the difference is considered to be significant, namely the statistical significance is achieved.
The abscissa in the graph of fig. 2A represents results of 10 days before, 10 days in the middle and 10 days after training, showing that the latency of 10 days in the middle of training is significantly reduced (P < 0.01) compared to the latency of 10 days before training, and the latency of 10 days after training is significantly lower than the latency of 10 days before and 10 days in the middle (P < 0.01), and the results of fig. 2B show that the latency of 30 days of DBA/2 mice and C57BL/6 mice is not significantly different (P > 0.05), indicating that the spatial learning ability of DBA/2 mice is significantly improved to a level equivalent to that of C57BL/6 mice after 30 days of Morris water maze training.
3) Screening of genes related to improvement of DBA/2 mouse spatial learning ability through Morris water maze training
After training, mice of a D2 control group, a D2 control group, a B6 control group and a B6 control group are killed under anesthesia, hippocampal tissues are separated and combined into 3 samples, total RNA samples are extracted, a transcriptome sequencing technology is utilized to construct gene expression profiles of the hippocampal tissues of the four groups of mice, the gene expression level is counted by an FPKM method, differential expression genes of the hippocampal tissues of the mice of the D2 control group and the B6 control group, the D2 control group and the D2 control group are respectively screened, and the screening standard is as follows: the expression variation multiple is more than 1.2 times, and the corrected P value is less than 0.05. 60 genes which are common in two groups of differentially expressed genes are selected, and then Nr1D1 genes with the largest expression variation multiple between the D2 training group and the D2 control group are obtained from 10 genes with the highest expression quantity, wherein the genes are related to the improvement of the space learning capacity of the DBA/2 mouse by Morris water maze training. Two-by-two comparison of the expression levels of the Nr1D1 gene in the hippocampus of mice in three groups, namely a D2 control group, a D2 training group and a B6 training group, is carried out by adopting one-way anova, and when p is less than 0.05, the difference is considered to be significant, namely, the statistical significance is achieved.
The results in FIG. 3 show that the Nr1d1 gene was expressed in the hippocampus of DBA/2 mice in a very significant amount (foldchagne)>1.2, Padj<0.001) lower than that of C57BL/6 mice, indicating that low expression of the Nr1d1 gene is associated with low spatial learning ability in DBA/2 mice.
The results in fig. 4 show that, compared with the untrained control group DBA/2 mice, the spatial learning ability of the mice after training is obviously improved, the expression level of the hippocampal Nr1d1 gene is also greatly improved (P < 0.01), the expression level of the hippocampal Nr1d1 gene of the training group C57BL/6 mice is also greatly higher than that of the untrained DBA/2 mice (P < 0.01), and the expression level of the hippocampal Nr1d1 gene of the training group DBA/2 mice and the training group C57BL/6 mice is not significantly different (P > 0.05), which indicates that the expression level of the Nr1d1 gene is also significantly increased to be not different from that of the C57BL/6 mice along with the improvement of the spatial learning ability of the DBA/2 mice, and indicates that the change of the expression level of the Nr1d1 gene is related to the spatial learning ability of the mice after Morris water maze.
The research shows that the space learning capacity of DBA/2 mice is obviously lower than that of C57BL/6, and the space learning capacity is probably related to the low expression of Nr1d1 gene. The space learning ability is always continuously strengthened and repeated to finally stabilize, after 30 days of water maze training, the space learning ability of a DBA/2 mouse is improved to reach the level equivalent to that of a C57BL/6 mouse, meanwhile, compared with an untrained DBA/2 mouse, the average expression level of an Nr1d1 gene in the hippocampus of the trained DBA/2 mouse is increased to 26.23, which is obviously higher than that of 19.42 of the untrained DBA/2 mouse, and the expression difference in the hippocampus of the trained C57BL/6 (25.8) mouse is not obvious, which shows that the space learning ability of the Nr1d1 gene and the rrMois trained and improved by the water maze training has obvious correlation, and the space learning ability can be used as a molecular biology evaluation marker for training or medicine to improve the space learning ability effect of the mouse.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (8)
1. A method for training and improving the space learning ability of a mouse is characterized by comprising the following steps: the water maze is used for training the mice, the training period is more than 6, and the times are 1-2 times per day.
2. The method for training the ability to improve spatial learning of a mouse according to claim 1, wherein: the training period is 6-30 days.
3. The method for training the ability to improve spatial learning of a mouse according to claim 1, wherein: the training period is more than 30 days.
4. The method for training the spatial learning ability of a mouse according to any one of claims 1 to 3, wherein the method comprises the following steps: the mouse is a DBA/2 mouse or a C57BL/6 mouse.
5. The method for training the spatial learning ability of a mouse according to any one of claims 1 to 3, wherein the method comprises the following steps: the week age of the mice is 4-8 weeks old.
6. The method for training the ability to improve the spatial learning of a mouse according to claim 4, wherein the method comprises the following steps: and repeatedly measuring anova is adopted in the latency period from the front of the DBA/2 mouse experiment to the back of the experiment, and the latency period and the C57BL/6 mouse latency period are analyzed by adopting independent sample t test, so that the improvement effect evaluation of the space learning capacity of the DBA/2 mouse is realized.
7. The method for training the ability to improve the spatial learning of a mouse according to claim 4, wherein the method comprises the following steps: and counting the expression level of the Nr1d1 gene in the mouse hippocampus before and after the DBA/2 mouse experiment, and comparing the expression level with the expression level of the C57BL/6 mouse Nr1d1 gene in the mouse hippocampus to realize the evaluation of the improvement effect of the space learning capacity of the DBA/2 mouse.
8. The application of the mouse Nr1d1 gene in improving the space learning ability of mice.
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