JP7080459B2 - Equipment and evaluation methods for assessing animal behavior - Google Patents

Description

The present invention relates to equipment for evaluating the behavior of animals, particularly experimental animals, and a method for evaluating the behavior. More specifically, the present invention relates to equipment for evaluating the effect of the drug candidate substance on the experimental animal based on the change in the self-issued behavior of the animal, and a method for evaluating the effect of the drug candidate substance based on the change in the self-issued behavior.

Alzheimer's disease is a symbol of neurodegenerative disease whose main symptom is a decrease in memory, which is caused by the degeneration and disappearance of hippocampal neurons, which mainly control memory. Neuropathologically, the neurodegenerative region of Alzheimer's disease extends beyond the hippocampus to the entorhinal cortex (parahippocampal gyrus), hippocampal limb, Nucleus basalis of Meynert, and tonsillar nucleus. For this reason, the Papez memory circuit (hippocampus → brain arch → mammillary body → mammillary body thorax: Vicq d'Azyr bundle → prefrontal cortex → zonal gyrus → zonal bundle → hippocampus (Closed circuit) and Yakovlev emotional circuit (mammillary body → dorsomedial nucleus of the thorax (nucleus medialis dorsalis [MD]) → prefrontal cortex (prefrontal cortex posterior 1/4 cortex region) → peritoneal cortex → closed circuit of the mammillary body ) Is also impaired, and in addition to memory ability, it is a disease that causes emotional disorders and decreased thinking ability, and overall cognitive function is decreased (Non-Patent Document 1). For this reason, Alzheimer's disease is called dementia.

The number of patients with Alzheimer's disease has been increasing rapidly in Japan in recent years, and wandering, disappearance, traffic accidents, etc. caused by this disease are becoming social problems. It is estimated that there are about 2 million people with dementia in Japan, 60% of whom are diagnosed with Alzheimer's disease. In addition to the treatment for Alzheimer's disease, the patient needs long-term care support to care for the patient in the daily environment. Therefore, the current situation is that not only the patient himself but also the patient's family and the caregiver are forced to bear a great deal of physical, mental and financial burden. It is estimated that in 2045, one in two people over the age of 65 will have Alzheimer's disease. In other words, Alzheimer's disease is said to be the most financially costly disease in developed countries including Japan from the viewpoint of economic cost-effectiveness ratio, and is even said to be a national disease. Nevertheless, no effective therapeutic agent for Alzheimer's disease has been established even now.

With regard to the diagnosis of Alzheimer's disease, so-called non-invasive diagnostic methods have been established. Here, the meaning of "non-invasive" means that when a definitive diagnosis of the patient is Alzheimer's disease, a biopsy histological diagnosis from the brain is not performed when making a definitive diagnosis of a brain tumor patient. That is, the patient is given a certain intellectual task to learn it, and if it cannot be learned well, the physical invasive stimulus evaluation method of giving a thermal stimulus or an electrical stimulus is not implemented. Specifically, the Wechsler Adult Intelligence Scale (WAIS), which accurately evaluates the higher-order functions of patients with Alzheimer's disease at the time of examination, and the Hasegawa dementia, which is easier to perform. Two higher-order function evaluation methods have been established (rating scale-revised: HDR-R). The Wechsler intelligence test is judged mainly by evaluation based on two factors: intelligence quotient (IQ) and behavioral intelligence. The Hasegawa simple intelligence evaluation scale mainly evaluates with verbal intelligence. When Alzheimer's disease is diagnosed by these clinical evaluation systems, it is generally called clinical Alzheimer's disease.
An accurate final diagnosis of Alzheimer's disease is made with a histopathological evaluation. That is, there is no final diagnosis of Alzheimer's disease without histopathological evaluation. In other words, the definitive diagnosis of Alzheimer's disease is made by the histopathological diagnosis of the autopsy brain after the death of a clinical Alzheimer's disease patient.

On the other hand, in natural mice, there is no spontaneously-onset highly cognitively impaired mouse corresponding to human Alzheimer's disease. Therefore, the cognitive function evaluation of mice is basically based on the premise that mice can learn. Therefore, the conventional method for evaluating cognitive function in mice does not assume mice with severe cognitive dysfunction such as human Alzheimer's disease, and is not a method that can be carried out non-invasively. Cognitive function evaluation methods currently performed using model animals such as mice, specifically, Morris water maze (Non-Patent Document 2), hot plate test (Non-Patent Document 2), and avoidance test by electrical stimulation (Non-Patent Document 2). The evaluation method such as 2) is a method of evaluating the learning achievement of a mouse by applying an invasive stress to an individual mouse. Usually, such an evaluation method requires a large number of trials in order to ensure the accuracy of the evaluation. Therefore, in the cognitive function evaluation of mice, there is an increased risk of mouse death due to stress on individual mice.

At this stage, as model mice for human Alzheimer's disease, human amyloid precursor protein gene and human tau protein gene, and double transgenic mice in which each human mutant gene is introduced into mice are histopathologically closest to human Alzheimer's patients. .. The cost of this double transgenic mouse is very high, for example, about 1.85 million yen for 10 mice on the original line. In experiments using such expensive Alzheimer's disease model mice, it is usual to kill the model at regular intervals and perform a histopathological evaluation of the brain in order to make an accurate final diagnosis of the results. Therefore, it can be said that the experiment using the Alzheimer's disease model mouse is an extremely expensive experimental system.
Where the model mouse, which is histopathologically closest to human Alzheimer's disease, has already lost its spatial cognitive learning ability, the conventional learning-type mouse cognitive function evaluation method is based on the premise that the mouse can learn. It is not appropriate in terms of the fact that it is present and that it adds a great deal of stress that leads to fatal accidents. Moreover, the histopathological evaluation method in which a definitive diagnosis is obtained at regular intervals is not suitable in terms of the cost of the experiment. However, the development of new therapeutic agents for human Alzheimer's disease is an urgent issue.
Therefore, in order to evaluate cognitive function using model mice easily, quickly, and at low cost, it is necessary to establish a non-invasive and non-learning evaluation method for experimental animals.

Kato et al., Acta Neuropathol 77: 258-266, 1989. Crawley JN. What ’s wrong with my mouse ?: Behavioral phenotyping of transgenic and knockout mice. 2nd ed. Hoboken, NJ: John Wiley & Sons, 2007

In view of the above circumstances, the present invention is a method for evaluating the self-issued behavior of an experimental animal, which is a non-learning type and stress-free (non-invasive) method based only on the daily self-issued behavior of the experimental animal. I will provide a.
The present invention also provides equipment used for the above evaluation method.
Furthermore, the present invention provides a method for screening a compound that affects the self-issued behavior of an experimental animal.

The inventors have created a space (about the width of the body of the experimental animal) between the cage wall and the rearing cage of the experimental animal so that the experimental animal can pass through, and the equipment (the equipment that restricts the behavior of the mouse, the following). We have developed a facility in which food and water (hereinafter referred to as “attractants”) are installed at a position about 1.5 times the body width of the experimental animal from the “behavior-restricting tool”).
The inventors used mice (Alzheimer's disease model mice and normal littermate mice) as animals for observing behaviors (self-issued behaviors), recorded only the spontaneous daily behaviors of the mice for 10 minutes, and analyzed their behaviors. rice field. In performing the behavior analysis, Activity and Trajectory were used as the elements of the behavior evaluation.
In the above equipment developed by the inventors, between the area where the attractant is easily accessible (the place where food and water are installed and its vicinity; the "attractor zone") and the behavior restriction device and the breeding cage. Space (“comfortable zone”) is formed.

Behavioral analysis of mice in cages without behavioral restriction equipment showed no difference in activity and trajectory between the Alzheimer's disease model mouse group and the littermate group. On the other hand, in the cage equipped with the restriction device, the activity of the Alzheimer's disease modelus group was 48.37 ± 5.24 in the attractant zone and 17.09 ± 5.45 in the relief zone, and the activity ratio of the normal littermate mouse group was the attractant. Zone: 8.95 ± 8.60, relief zone: 56.53 ± 17.35.
That is, the Alzheimer's disease model mice had significantly higher activity in the attractant zone, and showed abnormal eating behavior and decreased alertness. Spatial cognitive impairment in which the activity in the relief zone, which is supposed to give a sense of security, is significantly lower in Alzheimer's disease model mice than in normal littermate mice, and the risk avoidance ability is reduced and the activity in the space where the body should be placed is low. Was admitted.
As described above, it has been clarified that non-learning non-stress type experimental animal behavior evaluation becomes possible by using the equipment developed by the inventors.

The present invention has been completed based on the above findings.
Therefore, the present invention is the following (1) to (12).
(1) Equipment for evaluating the behavior of a laboratory animal, which is provided with a compartment for accommodating the laboratory animal, and an attractant and a behavior restriction tool are installed in the compartment.
(2) The shortest distance between the partition of the compartment and the outer surface of the behavior-restricting device is about 1.0 to about 1.5 times the width of the body of the experimental animal. The equipment according to (1) above, in which the behavior restriction tool is installed.
(3) The shortest distance from the outer surface of the behavior restriction device to the outer surface of the attractant is about 1.5 times to about 2.0 times the width of the body of the experimental animal. The equipment according to (1) or (2) above, in which the attractant is installed.
(4) A detection means for detecting an experimental animal in the compartment, the video means for photographing the inside of the compartment and the video means are operated, and the image captured by the video means is acquired as a video signal. The equipment according to any one of (1) to (3) above, further comprising a detection means including a control means.
(5) A method for evaluating the presence or absence of abnormalities in the self-issued movement of an experimental animal using the equipment according to any one of (1) to (4) above, wherein an image of the experimental animal acquired by the control means is obtained. An evaluation method using the Activity (activity) and / or Trajectory (center of gravity trajectory) of the experimental animal as an index, which is obtained by analysis.
(6) A method for evaluating the presence or absence of cognitive dysfunction in the experimental animal by using the method for evaluating the presence or absence of abnormal self-issued behavior of the experimental animal according to the above (5).
(7) The evaluation method according to (6) above, which comprises the following steps (a) to (c).
(A) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal of the control,
(B) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the attractant obtained in the attractant zone obtained in step (b) by the attractant obtained in step (a). The experimental animal has cognitive function when it is significantly higher than the activity in the zone and / or when the activity in the safe zone obtained in step (b) is significantly lower than the activity in the safe zone obtained in step (a). Step (8) The evaluation method according to (6) above, which includes the following steps (a) to (c).
(A) A step of determining the degree of accumulation of Trajectory in the attractant zone and / or the safe zone of the experimental animal of the control.
(B) The step of determining the accumulation degree of the disease in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the accumulation degree of the trajectory in the attractant zone obtained in the step (b) is the step (a). ) Is higher than the degree of accumulation of Trajectory in the attractant zone and / or the degree of accumulation of Trajectory in the safe zone obtained in step (b) is higher than the degree of accumulation of Trajectory in the safe zone obtained in step (a). Step of evaluating that the experimental animal has cognitive dysfunction (9) The diseases showing the cognitive dysfunction are Alzheimer-type dementia, cerebrovascular dementia, Lewy body dementia, and manic-depressive disease. , The evaluation method according to any one of (6) to (8) above, which is any of schizophrenia, developmental disorder and intellectual disability based on the aftereffects of the disease.
(10) The method according to any one of (5) to (9) above, wherein the experimental animal is a rodent or a small primate.
(11) A method for screening a compound effective for treating a disease associated with self-issued dyskinesia by using the method according to any one of (5) to (10) above.
(12) The disease showing the self-issued dyskinesia is any of Alzheimer's dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disability, and intellectual disability based on the aftereffects of the disease. The method according to (11) above, characterized in that it is a disease.

According to the equipment and the evaluation method of the present invention, it is possible to carry out the self-issued movement of the experimental animal by a non-invasive and non-learning method simply, quickly and at low cost.

The equipment and evaluation method of the present invention can also be used for screening of therapeutic agents and the like for diseases that affect the spontaneous daily behavior of experimental animals.

The schematic diagram of the equipment which concerns on this invention. Reference numeral 10; compartment, reference numeral 11; attraction installation position, reference numeral 12: action restriction device installation position, reference numeral 20: detection means, reference numeral 21; video means, reference numeral 22; control means, reference numeral 23; signal line. Equipment according to the present invention used in the examples. A video camera for capturing the behavior of an experimental animal from above and a general-purpose computer for operating the video camera and acquiring images are connected. The shooting was done in a bright field of view. The layout of the food and water (attractant) box and the mouse toy (behavior restriction tool) in the compartment of the equipment used in the embodiment. A is a layout plan in a normal breeding cage (compartment) in which only a food and a water box are installed without installing a mouse toy (behavior restriction tool) (Open field condition; food and water (attractant) in the normal breeding cage). (Conditions for installing only). B is a layout drawing in a normal breeding cage in which a mouse toy (behavior restriction tool) is installed (with Toy condition; a condition in which a mouse toy (behavior restriction tool) is installed in an Open field condition). An explanatory diagram in which the bottom of the cage is divided into five according to the spatial difference that occurs under With Toy conditions for the quantitative quantification of activity. Using Smart 3.0 used in this analysis, the analysis fields were arbitrarily divided and the values for each division were compared for statistical processing. Image excerpted from behavioral analysis video of normal littermate mice under Open field conditions. Mice (A) that were diagonally distant from food and water moved toward food and water (B) and then toward food and water (C). After the move, they acted to be interested in food and water (D). In addition, it showed irregular behavior patterns such as returning to the position of (A) as well as near food and water. Behavioral analysis results of normal littermate mice under Open field conditions. Activity 3D graph (A) and Trajectory image (B). A is a 3D graph showing the amount of activity corresponding to the position in the normal rearing cage (hereinafter, the same in FIGS. 6A, 8A, 10A and 12A). Activity activity is high in two places, around the food and water, and near the normal breeding cage angle in the opposite direction away from the position. B is a top view of the trajectory of the mouse Trajectory moving in the normal rearing cage (hereinafter the same in FIGS. 6B, 8B, 10B and 12B), and Trajectory corresponds to Activity. It accumulated near the food and water and near the upper right corner in the normal breeding cage away from it. Image excerpted from behavioral analysis video of Alzheimer's disease model mouse under Open field conditions. In the case of normal littermate mice (Fig. 5A), the experiment was started from the same position (A). Alzheimer's disease model mice moved towards food and water (B) and approached food and water boxes (C). Similar to normal littermate mice, after migration, they behaved in an interest in food and water (D). Similar to normal littermate mice, they showed irregular behavior patterns such as returning to position (A). Behavioral analysis results of Alzheimer's disease model mice under Open field conditions. Activity 3D graph (A) and Trajectory image (B). Similar to normal littermate mice, activity activity is high in two places, around food and water, and near the normal rearing cage angle in the opposite direction away from that position (A). Trajectory was also accumulated in the vicinity of food and water and in the vicinity of the upper right corner in a normal rearing cage away from it, as in normal littermate mice (B). Image excerpted from behavioral analysis video of normal littermate mice with With Toy conditions. The experiment was started from the position between the food and water installation position and the mouse toy installation position. We checked food and water (A), but did not move in that direction, but moved to Zone 4 (safety zone) surrounded by mouse toys and normal breeding cage walls (B). After that, he stayed in Zone 4 (safety zone) for a long time and was active there (C and D). Behavioral analysis results of normal littermate mice with With Toy conditions. Image of Activity 3D graph (A) and Trajectory (B). The activity in Zone 4 was very high and decreased as it approached the vicinity of food and water (A). In addition, a large amount of Trajectory was accumulated at the position where the activity was high (B). Image excerpted from behavioral analysis video of Alzheimer's disease model mouse with With Toy condition. The experiment was started from the same position as the normal littermate mouse. Alzheimer's disease model mice confirmed food and water (A), moved in that direction (B), and performed behaviors showing strong interest such as feeding and smelling. After that, it was confirmed that they frequently appeared near the place where food and water were installed (C and D). Behavioral analysis results of Alzheimer's disease model mice with With Toy conditions. Image of Activity 3D graph (A) and Trajectory (B). Activity was very high in the vicinity of food and water, high in normal littermate mice and low in Zone 4 (safety zone) (A). Trajectory was accumulated in a circle along the circumference of the mouse toy, and no accumulation was normally observed near the cage wall (safety zone) (B). A graph showing the ratio (Activity%) of Activity in each Zone under the With Toy condition. The percentage of activity (Activity%) of Alzheimer's disease model mice (black graph: 3) and normal littermate mice (white graph: 4) in each of the five zones was graphed. A Mann-Whitney U test showed a significant difference of * p <0.05 between Zone 2 and Zone 4. The correspondence between the results of Activity% (Fig. 13) and Trajectory (Fig. 12B) of Alzheimer's disease model mice and the results of histopathological analysis is shown. Effect of compound K that can suppress the formation of amyloid plaque and neurofibrillary tangles. When compound K was administered to Alzheimer's disease model mice, what kind of changes occurred in the activity (upper row) and trajectory (lower row) of the mice were investigated. The image pattern of the activity (upper row) of the Alzheimer's disease model mouse to which the compound K was administered showed an intermediate type of each image pattern of the Alzheimer's disease model mouse to which the placebo was administered and the normal littermate mouse to which the placebo was administered. The results of histopathological analysis of the brain of Alzheimer's disease model mice to which Compound K was administered are shown. We compared the formation of amyloid plaque and changes in neurofibrils in the brains of placebo-administered Alzheimer's disease mice and normal littermate mice, and compound K-administered Alzheimer's disease model mice. Placebo-treated Alzheimer's disease mice show a large number of amyloid plaques and neurofibrillary tangles. Neither structure was observed in normal littermate mice treated with placebo. The formation of amyloid plaque and neurofibrillary tangles was significantly suppressed in the Alzheimer's disease model mice treated with Compound K as compared with the Alzheimer's disease model mice treated with placebo.

A first embodiment of the present invention is a device for evaluating the behavior of a laboratory animal, which comprises a compartment for accommodating the laboratory animal, in which an attractant and a behavior restriction device are installed. It is a facility that is available.
The equipment according to the first embodiment includes a compartment (reference numeral 10 in FIG. 1) capable of accommodating a laboratory animal. Here, the "compartment" is a space in which an experimental animal can be accommodated (entered) and the surroundings are surrounded by a partition or the like so that the experimental animal does not escape from the compartment. Typical examples of compartments include, but are not limited to, cages and cages for animal rearing. The material of the compartment partition may be any material as long as it is not easily destroyed by the laboratory animal contained therein, and may be, for example, metal, plastic, glass, wood or the like. Further, the partition may have any shape such as a net or a plate. The shape of the compartment (a partitioned space, the shape of the space seen from above the facility) is also not limited and may be, for example, a square typical of cages for domestic animals. Alternatively, it may have any shape such as a circle, an ellipse, a triangle, and a rhombus.
The size of the compartment (the bottom area surrounded by the partition) is particularly limited as long as the experimental animal is free to move and is large enough to perceive attractants and behavior-restricting devices during action. Not done. As an experimental animal, for example, when a mouse is used and the shape of the compartment is a quadrangle (square, rectangle, etc.), the length of each side is, for example, about 20 cm to about 100 cm, preferably about. It is about 40 cm to about 70 cm. The height of the compartment may be such that the experimental animal cannot escape. For example, when using a mouse, the height may be, for example, about 20 cm to about 60 cm, preferably about 25 cm to about 50 cm. ..

The "attractant" (reference numeral 11 in FIG. 1) installed in the compartment of the first embodiment is an object that the experimental animal instinctively prefers, or an object that influences the instinct of the experimental animal and attracts the animal. Therefore, for example, in addition to food and water, it may be a dependent drug, a pheromone that attracts an experimental animal to be used, or the like. As the attractant, only the attractant may be installed in the compartment, but if necessary, it may be placed in a suitable container and installed in the compartment.
Further, the "behavior-restricting device" (reference numeral 12 in FIG. 1) installed in the compartment of the first embodiment is an object that hinders the behavior of the experimental animal. For example, it is preferable that the animal does not easily get over the animal, but that the animal's behavior is restricted by changing the traveling direction of the animal. The action restricting tool is preferably a three-dimensional object, and the shape is not particularly limited, and may be any shape such as a spherical shape, a square shape, or a flat plate shape (tuitate shape).
The size (volume) of the behavior restriction device is preferably larger than the volume of the body of the experimental animal to be used, for example, about 1 to about 2,500 times, more preferably about 10 times to the volume of the body of the experimental animal to be used. It is about 1,000 times. When, for example, a mouse is used as an experimental animal, the size of the limiting tool is preferably about 10 to 1,000 times the volume of the mouse.

It is desirable that the behavior-restricting equipment should be installed in the compartment with a space between the compartment partition and the behavior-restricting equipment so that the experimental animal can place it. For example, it is preferable that the shortest distance between the partition of the compartment and the outer surface of the behavior-restricting device is about 1.0 to about 1.5 times the width of the body of the experimental animal to be used.
When a mouse is used as an experimental animal, the shortest distance between the partition of the compartment and the outer surface of the behavior-restricting device is preferably, for example, about 2 cm.
In this specification, the space area created between the partition of the compartment and the behavior restriction tool is referred to as a "safety zone".

The installation position of the attractant in the compartment is preferably a position where the experimental animal cannot reach the attractant unless it moves from the above-mentioned "safety zone", and the experimental animal can perceive the attractant from the "safety zone". .. For example, the distance from the outer surface of the behavior restriction device to the outer surface of the attractant, and the shortest distance is preferably about 1.5 to about 3.0 times the width of the body of the experimental animal to be used, preferably about 1.5. About times to about 2.0 times is more preferable. Here, the outer surface of the attractant is the outer surface of the attractant when only the attractant is installed in the compartment, and the outside including the container when the attractant is placed in a container or the like. It is the surface.
When a mouse is used as an experimental animal, it is preferable that the shortest distance from the outer surface of the behavior restriction tool to the outer surface of the attractant is, for example, about 4 to 80 cm.
In this specification, the range in which the experimental animal can come into contact with the attractant without moving is referred to as an "attractant zone".

The equipment according to the first embodiment may further include a detection means 20 for detecting the behavior of the laboratory animal in the compartment (FIG. 1). The detection means 10 includes a video means 21 such as a video camera for photographing an experimental animal, a control means 23 such as a computer that operates the video means and acquires an image captured by the video means as a video signal, and a video. It is composed of a signal line 22 for transmitting and receiving a signal between the means and the control means. Here, the computer as the control means not only receives the video signal of the experimental animal and displays the continuous video of the experimental animal on the display of the computer, but also incorporates software for analyzing the continuous video. You may be.

The second embodiment of the present invention is a method for evaluating the presence or absence of abnormalities in the self-issued movement of an experimental animal using the equipment according to the first embodiment, and is a control means provided in the first embodiment. This is an evaluation method using the Activity (activity) and / or Trajectory (center of gravity trajectory) of the experimental animal as an index, which is obtained by analyzing the image of the experimental animal acquired by.
Self-issued behavior is a general term for behaviors related to life support based on instinct, such as movement, climbing, tail chasing, standing up, grooming, and eating behavior.
Here, Activity and Trajectory can be obtained by analyzing the images of experimental animals. The time for photographing the experimental animal varies depending on the experimental animal to be used, and may be any time required for photographing a series of self-issued movements. For example, it may be several minutes to several hours (or several days), and particularly when using a small rodent such as a mouse, it is preferably within one hour, more preferably about ten and several minutes. ..
Activity is the sum of the self-issued movements, and is, for example, the amount of variation (for example, the position of the center of gravity of the experimental animal, the body surface such as the head and the tail) of a specific body surface point of the experimental animal with respect to the compartment (for example). , The moving speed of a specific body surface point, the moving distance, etc.) can be quantified. Activity quantification is based on information-based analysis software that tracks laboratory animals (eg, Smart 3.0, Panlab SLU, Barcelona, Spain; PhenoScan series, Prime Tech Co., Ltd., Tokyo, Japan; EthoVision XT, Netherlands Information Technology, Vergeningen. , Netherlands ,; CompACT series, Muromachi Kikai Co., Ltd., Tokyo, Japan; ANY-maze, Stoelting Co., Wooddale, Illinois, USA; ABDigitizer, Intelligent Information Systems Co., Ltd., Kyoto, Japan, etc.) It can be easily carried out at.

Trajectory tracks experimental animals that perform self-issued movements and draws their trajectories as lines. In areas where many spontaneous movements of experimental animals are observed, lines depicting Trajectory accumulate (the number of trajectory lines increases). The degree of accumulation of Trajectory in a certain region can be determined, for example, by counting the number of lines drawn on the trajectory of the center of gravity of the experimental animal in the region. For example, FIG. 10B tracks an experimental animal and uses analysis software to represent the locus of the center of gravity in the compartment as a line. Since the number of trajectory lines in the safe zone is larger than the number of trajectory lines in the other zones, it can be determined that the degree of accumulation of Trajectory in the safe zone is higher than that in the other zones. Trajectory is an information-based analysis software that tracks laboratory animals (eg Smart 3.0, Panlab S.L.U., Barcelona, Spain; PhenoScan series, Prime Tech Co., Ltd., Tokyo, Japan; EthoVision XT, Nordus Information Technology, Vergeningen, Netherlands. ; CompACT series, Muromachi Kikai Co., Ltd., Tokyo, Japan; ANY-maze, Stoelting Co., Wooddale, Illinois, USA; ABDigitizer, Intelligent Information Systems Co., Ltd., Kyoto, Japan; Imagepower TrackingEye, Fujitsu Kyushu System Service Co., Ltd. , Fukuoka, Japan, etc.) can be used to easily draw lines. By counting the number of trajectory lines at any location in the compartment or visually estimating the degree of trajectory accumulation, the degree of Trajectory accumulation at any location can be determined.

In the evaluation method according to the second embodiment of the present invention, the Activity and / or Trajectory of the experimental animal to be evaluated is compared with the Activity and / or Trajectory of the control experimental animal (experimental animal whose spontaneous movement is normal). This is a method of evaluating (or determining) that the experimental animal to be evaluated has some kind of spontaneous dyskinesia when a result different from that of the control animal is obtained. When comparing Activities, Activities can be easily compared by quantifying them as described above. In addition, for Trajectory, the degree of accumulation in a specific region in the movement trajectory (trajectory of the center of gravity of the animal) of each experimental animal (control and evaluation target animal) can be evaluated and compared.

The above-mentioned abnormalities in self-issued behavior are also observed when cognitive dysfunction (for example, dementia, manic depression, schizophrenia, developmental disability, and intellectual disability based on sequelae of illness, etc.) develops.
Therefore, the third embodiment of the present invention is a method for evaluating the presence or absence of cognitive dysfunction in the experimental animal by using the method for evaluating the presence or absence of abnormalities in the self-issued behavior of the experimental animal according to the second embodiment. Is. When the self-issued dyskinesia characteristic of cognitive impairment is observed in the animal to be evaluated by using the method for evaluating the self-issued dyskinesia of the experimental animal according to the second embodiment, the experimental animal to be evaluated has cognitive function. It can be evaluated (or judged) as suspected of having a disorder.
When Activity is used as an evaluation index for self-issued behavioral disorders, the presence or absence of cognitive dysfunction in experimental animals can be evaluated (determined) by performing the following steps (a) to (c).
(A) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal of the control,
(B) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the attractant obtained in the attractant zone obtained in step (b). The experimental animal has cognitive impairment when it is significantly higher than the activity in the zone and / or when the activity in the safe zone obtained in step (b) is significantly lower than the activity in the safe zone obtained in step (a). Process to evaluate as being

When Trajectory is used as an evaluation index for self-issued behavioral disorders, the presence or absence of cognitive dysfunction in experimental animals can be evaluated (determined) by performing the following steps (a) to (c).
(A) A step of determining the degree of accumulation of Trajectory in the attractant zone and / or the safe zone of the experimental animal of the control.
(B) The step of determining the degree of accumulation of Trajectory in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the degree of accumulation of Trajectory in the attractant zone obtained in step (b) is the step (a). ) Higher than the accumulation of Trajectory in the attractant zone and / or the accumulation of Trajectory in the safe zone obtained in step (b) is higher than the accumulation of Trajectory in the safe zone obtained in step (a). Step to evaluate that the experimental animal has cognitive dysfunction when the value is low In the evaluation of the presence or absence of cognitive dysfunction in the experimental animal, both Activity and Trajectory may be used as an index for evaluation.

The evaluation method according to the third embodiment is, for example, Alzheimer's dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disability, and intellectual disability based on the aftereffects of the disease. It can be used to evaluate self-issued schizophrenia in laboratory animals caused by. For example, as shown in the examples, when a disease model animal such as Alzheimer's disease is present, the abnormal behavior characteristic of the disease (for example, by comparing the spontaneous behavior of the disease model animal and the normal animal) is compared. In the case of Alzheimer-type dementia, the activity is high in the attractant zone and the accumulation of Trajectory is high).

The evaluation methods according to the second and third embodiments are for evaluating the presence or absence of abnormalities in self-issued behavior and the presence or absence of cognitive dysfunction. Therefore, it can be used for screening of compounds effective for the treatment and / or prevention of diseases showing abnormal self-issued behavior, for example, diseases associated with cognitive dysfunction.
Candidate compounds for the treatment (or prevention) of the disease were administered to disease model animals showing self-issued behavioral abnormalities evaluated by the evaluation methods according to the second and third embodiments, and the self-issued behavioral abnormalities were improved. If the behavior is similar to that of a normal experimental animal, or if the behavior is intermediate between the behavior of a disease model animal and the behavior of a normal experimental animal, the candidate compound is selected. , Can be determined to be effective in treating the disease.
In particular, when using model animals with pathological characteristics characteristic of the disease, such as Alzheimer-type dementia model mice (for example, Tg (APPSWE) 2576Kha Tg (Prnp-MAPT * P301L) JNPL3HImc mice), the book It is possible to associate (associate) the abnormalities of self-issued motion evaluated by the embodiment of the present invention with pathological features (see Examples), and only by the evaluation method of the present invention (pathological diagnosis). Simple and rapid primary screening of candidate compounds is possible.

The experimental animals used in the embodiments of the present invention are not particularly limited, but are not limited to small animals such as rodents (eg, mice, rats, hamsters, guinea pigs, tsupai, etc.), as well as small primates (common marmosets). Etc.) are preferable.

The disclosures of all references cited herein are incorporated herein by reference in their entirety. Also, throughout the specification, when the singular words "a", "an", and "the" are included, not only the singular but also the plural, unless the context clearly indicates otherwise. It shall include things.
Hereinafter, the present invention will be described with reference to examples, but the examples are merely examples of embodiments of the present invention and do not limit the scope of the present invention.

Materials and methods 1. Mouse breeding cage (compartment) used for analysis system (equipment according to the present invention)
Normal mouse breeding cage (product number: KE-3101, Theobbit Co., Ltd., Tokyo, Japan) and mouse food (trade name: CE-2, Japan Claire Co., Ltd., Tokyo, Japan) 2 pieces (1 piece) : 3.5g) and mouse drinking water (water from tap water, which is human drinking water, filtered using a filter with a filtration accuracy of 0.5 μm (manufacturer: Aion, product name: KANEFIEL, model number: FD-005) (Used) A box made of 113 mm x 81 mm x 15 mm (height) paper on which a glass container (trade name: BT-500 lid, Matsunami Glass Industry Co., Ltd., Wada, Osaka, Japan) filled with about 5 ml is placed. A food / water box) (attractor) was placed, and a mouse toy (trade name: K3570 / Mouse Igloo (Blue), Bio Serv, Flemington, New Jersey, USA) (behavior restriction tool) was placed. This cage is lined with a floor bed (trade name: Pure Chip, Shimizu Laboratory Materials Co., Ltd., Kyoto, Japan) with a thickness of about 1 cm, and the food, water box, and mouse toys are placed on it.

2. 2. Box device for photography used for analysis system In addition to the mouse breeding cage for analysis, a box device for photography (outer dimensions = length: 240 mm, width: 351 mm, height: 700 mm, thickness: 10 mm made of polycarbonate) was installed. .. Two holes were made in the top plate of the Box device for shooting to attach the camera (trade name: Kinect for Windows sensor L6M-00005, manufacturer: Microsoft Corporation, Raymond, Washington, USA), and the camera was fitted. In order to stabilize the box device for photography, aluminum weights (weight per one: 500 g) were attached below the four sides of the box device for photography. This box device for photography was used by covering it with a mouse breeding cage for analysis, which is usually a breeding cage for mice.

3. 3. Peripheral equipment used for analysis system General-purpose personal computer used ([Model: ASPIRE V3-571-H54D / K, Manufacturing company: Acer Incorporated, Shinboku, Republic of China], [OS: Windows 7 Home Premium, Manufacturing company: Microsoft Corporation , Raymond, Washington, USA]), modified the software for capturing camera data (trade name: Microsoft Visual Studio, manufacturer: Microsoft Corporation, Raymond, Washington, USA) to prepare the shooting environment. In this system, the camera is automatically activated by the operation from the personal computer (personal computer), and it is possible to shoot for 10 minutes. The recorded video was analyzed using analysis software (trade name: Smart 3.0, Panlab SLU, Barcelona, Spain). The moving image shooting recording in this embodiment is 10 minutes after the mouse is placed, and the total shooting capacity is set to be about 20MB per recording. All of this data was set to be automatically recorded on a personal computer (Fig. 2). The peripheral devices used in the analysis system were installed as shown in FIG.
The mouse breeding cage for analysis is installed so that the short side of the normal mouse breeding cage overlaps with the long side of the food and water box on the upper left when viewed from above, and the mouse toy is 2.5 cm to 3.5 cm from the right end of the breeding cage. It was installed so that it would be in between. Although the glass container for water was installed inside the box, it is desirable to install it as close as possible to the long side of the breeding cage. The bait was placed near the short side of the bait / water box on the opposite side of the position where the glass container for water was placed (Figs. 3 and 4).

4. Zone setting in a normal breeding cage (With Toy) with mouse toys installed
In the normal breeding cage under the With Toy condition, five Zones (compartments) were set as shown in FIG. Zone2: Zone (feed zone; attractant zone) where mice can eat food, Zone3: Zone where toys are placed, Zone4: Zone (Comfortable Zone: comfortable and secure for mice) which seems to correspond to a burrow in nature Zone, Zone 5: Not close enough to eat food, but showing interest in food and water, which is thought to be the most calming for normal mice. Zone, Zone1: The above Zone2 to Zone5 are subtracted from the total bottom surface (Total). In this embodiment, the settings (= Detection Settings) are optimized according to the difference in color between the color of the mouse hair and the color of the mouse toy. The detailed setting values of Smart 3.0 are as follows. If the mouse toy is red and the hair is gray or black: Brightness & Contrast-Brightness: 255, Contrast: 176, Use special lighting conditions for this zone: no check the box, Track Detection-Detection = Threshold: 87, Erosion: 3, Subjects: 1, Activity Detection = 8. If the toy is blue and the hair is gray or black: Brightness & Contrast-Brightness: 231, Contrast: 231, Use special lighting conditions for this zone: check the box, zone: Zone3 Brightness: 101 & Contrast: 115, Track Detection- Detection = Threshold: 87, Erosion: 3, Subjects: 1, Activity Detection = 8.

5. As experimental animal Alzheimer's disease model mice, strain: Tg (APPSWE) 2576KhaTg (Pmp-MAPT * P301L) JNPL3HImc mice (Taconic, New York, USA) were used as normal target mice, and littermate mice of the mice (Pmp-MAPT * P301L). Taconic, New York, USA) were used.
In Tg (APPSWE) 2576KhaTg (Pmp-MAPT * P301L) JNPL3HImc mice, the human mutant APP gene (Sweden) in which the 670th lysine of the human amyloid precursor protein (APP) gene was point-mutated to methionine and the 671st asparagine was point-mutated to leucine. It is a double transgenic mouse produced by introducing a human tau mutation gene (gene) that causes hereditary Alzheimer's disease) and a human tau mutation gene in which the 301st proline of the human tau protein gene is point-mutated to leucine. This Alzheimer's disease model mouse expresses amyloid plaque and neurofibrils at about 1 year of age.

6. Statistical analysis method Quantitative values of Alzheimer's disease model mice and normal littermate mice between Zones 1 to 5 when a mouse toy is installed are shown by mean ± standard deviation. All statistical analyzes were performed using Macintosh Software's StatView: Version 5.0 (SAS Inc., Cary, North Carolina, USA). The Mann-Whitney U test was used for the significance test, and it was considered that there was a statistically significant difference when the risk rate was less than p <0.05.

7. Histopathological analysis Compound K and placebo (solvent of compound K), which can suppress the formation of senile plaques and neurofibrils, were administered to experimental animals. As for the method of oral administration, the volume was accurately measured with a plastic syringe, and a gastric sonde for mice was directly connected to the plastic syringe for reliable oral and transesophageal administration.
As experimental animals, in particular, a gene having a Swedish mutation in the gene encoding the 695 amino acid of the amyloid β (Aβ) precursor protein, and the 301st proline of the human tau protein gene were found in human Alzheimer's disease type dementia model mice. Characteristic of the disease, such as Tg (APPSWE) 2576KhaTg (Prnp-MAPT * P301L) JNPL3HImc strain Alzheimer's disease model double transgenic mouse, which highly expresses both human genes of the mutant gene mutated to leucine (P301L). Use model animals with different histopathological characteristics. Similarly, normal littermate mice were also used.

The identification of amyloid plaque and neurofibrils appearing in the experimental animals was performed by histochemical staining and immunohistochemical staining. As histochemical staining, for example, hematoxylin and eosin (HE) staining was performed. For immunohistochemical staining, we detected amyloid β protein, which is the core protein of amyloid plaque, and phosphorylated tau protein, which is the core protein of neurofibrillary tangles. For example, as a method for detecting amyloid β protein, an amyloid β protein immunohistochemical staining kit (Code No.299-56701, Wako Pure Chemical Industries, Ltd., Osaka) was used. The anti-amyloid β protein (1-40) mouse monoclonal antibody (clone No. BA27) in the kit was used for the detection of Aβ40. The anti-amyloid β-protein (1-42) mouse monoclonal antibody (clone No. BC05) in the kit was used for the detection of Aβ42. Finally, visualization was performed using 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dako, Glostrup, Denmark) as a color former. In addition, as a method for detecting phosphorylated tau protein, the following primary antibody was used in combination with the ABC (avidin-biotin-immunoperoxidase complex) method. As the primary antibody, an anti-phosphorylated tau protein (PHF-tau) mouse monoclonal antibody (clone: AT8, Innogenetics: currently company name Fujirebio Co., Ltd., Tokyo) was used. The ABC kit used was the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Finally, DAB was visualized as a color former. Optical microscope equipped with 3CCD digital camera system (FX380: Olympus) equipped with image image analysis software (FLVFS-LS Ver. 1.12: Olympus, Tokyo) for each stained specimen of HE staining, Aβ40 immunostaining, Aβ42 immunostaining, and AT8 immunostaining. Staining was performed with (BX41: Olympus), and image analysis was performed along with photography with the device.
The above experiments were carried out with the approval of the Animal Experiment Committee of Tottori University.

Result 1. As a result of installing only food and water box in the breeding cage (compartment) (Open field condition) Analysis under the Open field condition without installing the mouse toy (behavior restriction tool) is performed in the normal breeding cage, long side: 113 mm × Short side: 81 mm x height: 15 mm box containing about 5 ml of drinking water for mice and 2 baits of 3.5 g per piece (food / water box) (attracted object), upper left when viewed from above It was analyzed while it was placed in a corner.

1-1. Behavioral analysis results of normal littermate mice under Open field conditions.
Regarding the behavior of normal littermate mice, the experiment was started from the position opposite to the food and water installation position (Fig. 5A). First, they approached the vicinity of food and water, which are strong instinctive stimulants (Fig. 5B). When they reached the vicinity of the bait and water, they sniffed the bait and showed interest (Figs. 5C and D). However, this state does not continue, and it moves away from it in a short time, moves in the direction opposite to the position where the food and water are installed, and then reverses and moves again in the vicinity of the food and water. It did not show a constant behavioral inclination pattern.
FIG. 6 shows Activity and Trajectory in a 10-minute analysis of normal littermate mice under Open field conditions. Activity was high in two places, around the bait and near the upper right corner in the opposite direction away from it (Fig. 6A). In addition, Trajectory was accumulated near the upper right corner of the cage on the opposite side of the food and water installation position so as to correspond to Activity (Fig. 6B).

1-2. Behavioral analysis results of Alzheimer's disease model mice under Open field conditions.
Images showing the behavior of Alzheimer's disease model mice were started from a position opposite to the food and water placement position as in the case of normal littermate mice for easy comparison (FIG. 7A). Alzheimer's disease model mice approached the instinctive stimulants, food and water (Fig. 7B), and showed interest such as sniffing food and water (Fig. 7C and D). However, in a relatively short time, it once separated from the food and water and moved in the direction opposite to the position where the food and water were installed. From the position after the movement, it moved again in the direction of the position where the food and water are installed. By repeating such behaviors, they did not show a certain behavior pattern.
Figure 8 shows the activity and trajectory in a 10-minute analysis of Alzheimer's disease model mice under open field conditions. It is very similar to the Trajectory pattern of normal littermate mice, and high activity was observed in two places, near the food and water placement and near the short side of the cage in the direction opposite to the food and water placement position (Fig.). 8A). In addition, the Trajectory accumulated a large amount of Trajectory in the vicinity of food and water and near the short side of the cage in the opposite direction to the normal littermate mice (FIG. 8B).

1-3. Statistical comparison results between the normal littermate mouse group and the Alzheimer's disease model mouse group under Open field conditions.
As described above, even under the Open field condition, five zones were virtually set and examined in the same manner as when the mouse toy (behavior restriction tool) was installed (With Toy condition).
The analysis results of the activity of the normal littermate mouse group (n = 4 mice) under the Open field condition are Zone1 = 25.58 ± 3.06 (mean ± standard deviation), Zone2 = 27.94 ± 3.66, Zone3 = 18.14 ± 3.76, Zone4 = 18.39. It was ± 1.73 and Zone5 = 9.96 ± 0.54. On the other hand, the analysis results of the activity of the Alzheimer's disease model mouse group (n = 3 mice) are Zone1 = 22.32 ± 2.28, Zone2 = 29.94 ± 10.83, Zone3 = 24.55 ± 3.53, Zone4 = 17.80 ± 10.44, Zone5 = 5.37 ± 1.75. there were.
When the significant difference between the normal littermate mouse group and the Alzheimer's disease model mouse group was examined, no significant difference was observed between the two groups, Zone1, Zone2, Zone3, Zone4, and Zone5.
In addition, there was no difference in the accumulation results of Trajectory between the normal littermate mouse group and the Alzheimer's disease model mouse group (FIGS. 6B and 8B).

2. 2. As a result of the additional installation of a mouse toy (behavior restriction tool) in the Open field condition (With Toy condition), the food and water (attractants) were installed in the same position as in the Open field condition. That is, it was placed at the upper left corner when viewed from above. Mouse toys (behavioral restriction tools) were installed so as to be between 2.5 cm and 3.5 cm from the right end of the breeding cage.

2-1. Behavioral analysis results of normal littermate mice under With Toy conditions.
For normal littermate mice, the experiment was started from the intermediate position between the position where the instinct stimulant food and water were installed and the position where the mouse toy was installed. Normal littermate mice looked in the direction of food and water and recognized that they were food and water (Fig. 9A). However, although normal littermate mice recognized food and water, they moved in the opposite direction to their placement position (Fig. 9B), and the mouse toys and normal rearing cages on the opposite side of the food and water placement position. I moved to the area between the walls (Zone 4 (Comfortable zone)) and put myself there (Figs. 9C and D).
Although the experiments in this example were performed in the bright field, Zone 4 can be assumed to be the most homologous place (safety zone) to the burrow that protects the body from the danger of the outside world in the bright field of normal wild mice. Therefore, it is considered that the normal littermate mice were placed in Zone 4.
FIG. 10 shows Activity and Trajectory in a 10-minute analysis of normal littermate mice.
From the 10-minute activity analysis results (Fig. 10A) of normal littermate mice, it was found that the activity was highest in Zone 4 (safety zone) and low in Zone 2 (attracting zone) where food and water boxes were installed. ..
In the 10-minute accumulated image of Trajectory, the degree of accumulation of Trajectory was the highest in Zone 4 (safety zone) as well as the result of Activity (Fig. 10B). In addition, from the photographed image, the normal littermate mouse moves along the wall surface of the normal breeding cage, and is mounted against the wall of the normal breeding cage in Zone 4 (safety zone) (the mouse raises its forelimbs to the wall and extends its hind limbs). It was confirmed that the so-called rising search behavior) was performed. The behavior of such normal littermate mice corresponds to the integrated images of Trajectory.

2-2. Behavioral analysis results of Alzheimer's disease model mice under With Toy conditions.
Behavioral analysis of Alzheimer's disease model mice was also started from an intermediate position between the food and water installation positions and the mouse toy installation positions, as in the normal littermate mice (FIG. 11A). Alzheimer's disease model mice immediately moved in the direction of food and water (Fig. 11B) and placed themselves in Zone 2 (attracting zone) where the food and water box were placed. From a behavioral point of view, the behavior of sniffing the food and eating the food was the first to be performed (FIGS. 10C and D). Eating behavior in the bright field corresponds to the most dangerous behavioral period when wild animals are easily attacked because attention is paid to food and water in the natural world. Nevertheless, it was found that this long-term abnormality in feeding behavior was observed in Alzheimer's disease model mice from the early stage of imaging.
The results of 10-minute activity and trajectory of Alzheimer's disease model mice are shown in FIG. Activity was high in Zone 2 (attracting zone), where food and water were installed, and low in Zone 4 (safety zone), which is supposed to correspond to a burrow in nature (Fig. 12A).
In addition, Trajectory showed a high degree of accumulation in Zone2 (attracting zone), and showed an image similar to that of a mouse toy in Zone4 (safety zone) (Fig. 12B). No accumulation of Trajectory was observed along the wall of the normal rearing cage as seen in normal littermate mice.

2-3. Statistical comparison of activity between normal littermate mouse group and Alzheimer's disease model mouse group under Toy condition.
Analysis results of With Toy conditions in the normal littermate mouse group (n = 4) are Zone1 = 25.63 ± 9.56, Zone2 (attractor zone) = 8.95 ± 8.60, Zone3 = 3.09 ± 1.13, Zone4 (safety zone) = 56.53 ± It was 17.35, Zone5 = 5.79 ± 3.48.
The analysis results of With Toy in the Alzheimer's disease model mouse group (n = 3) are Zone1 = 24.35 ± 1.46, Zone2 (attractor zone) = 48.37 ± 5.24, Zone3 = 3.55 ± 2.03, Zone4 (safety zone) = 17.09 ± 5.45. , Zone5 = 6.65 ± 4.20.
In Zone 2 (attractant zone), the activity of the Alzheimer's disease model mouse group was significantly higher than that of the normal littermate mouse group (p <0.05). In Zone 4, the activity of the Alzheimer's disease model mouse group was significantly lower than that of the normal littermate mouse group (p <0.05). In Zone1, Zone3, and Zone5, there was no significant difference between the normal littermate mouse group and the Alzheimer's disease model mouse group.
The above results are shown in FIG.

2-4. Comparison of Trajectory between normal littermate mouse group and Alzheimer's disease model mouse group under Toy condition.
The comparison of Trajectory was made based on the comparison of the degree of accumulation of Trajectory lines in one mouse for 10 minutes.
In the normal littermate mouse group, a large amount of Trajectory in Zone 4 (safety zone) was accumulated on the wall surface of the normal rearing cage (Fig. 10B). This result indicates that the same behavioral pattern as that of naturally occurring wild mice moving along the wall surface is also observed in normal littermate mice. In addition, the fact that normal littermate mice mounted on the wall surface, which is one of the exploratory behaviors, was also consistent with the typical behavior pattern of wild mice.
In contrast, the Zone 4 Trajectory in the Alzheimer's disease model mouse group did not show the accumulation of drawn lines in contact with the wall surface of the cage, but accumulated around the mouse toy (Fig. 12B), and was a normal littermate mouse. It was different from the Trajectory drawn by. That is, in the Alzheimer's disease model mice, a decrease in alert behaviors (movement along the wall surface, mounting search behavior, etc.) exhibited by normal littermate mice was observed. This result indicates that the Trajectory accumulation degree of normal littermate mice and Alzheimer's disease model mice can be distinguished by simply comparing the results of Zone 4 (safety zone).
In addition, there was a difference between the normal littermate mouse group and the Alzheimer's disease model mouse group in the analysis results in Zone 2 (attractant zone) where food and water were installed. That is, the Trajectory of the Alzheimer's disease model mouse group showed strong accumulation in Zone 2 (attractant zone) as compared with the normal littermate mouse group. There was no clear difference in the degree of Trajectory accumulation in the other zones between the normal littermate mouse group and the Alzheimer's disease model mouse group.

2-5. Correspondence between Activity% and Trajectory results of Alzheimer's disease model mice and histopathological analysis results.
The histopathological images of amyloid plaque and neurofibrils on the hippocampal secant of Alzheimer's disease model mice showing the activity% of With Toy shown in FIG. 13 and the accumulation results of Trajectory as shown in FIG. 12B were plotted (FIG. 14). .. As a result, as shown by the black graph in FIG. 13, in the Alzheimer's disease model mice in which the activity% in zone 2 was high and the strong accumulation of trajectory in zone 2 was observed, the hippocampus and its surroundings, which control memory, were observed. A large number of neurofibrils were found in the amyloid plaque and near the amygdala, which is considered to be the center of emotion.
From the above, it was clarified from the histopathological analysis that the Alzheimer's disease model mice exhibiting the symptoms of Alzheimer's disease showed a high activity% value and a strong accumulation of Trajectory in Zone 2.

2-6. Effect of compound K administration that can suppress the formation of amyloid plaque and the formation of neurofibrils on activity and trajectory Candidate compound K or placebo that suppresses the formation of amyloid plaque and the formation of neurofibrils was added to Alzheimer's disease model mice. In addition, placebo was administered to normal littermate mice for 365 days or longer, and then the presence or absence of abnormal self-issued behavior of these mice was evaluated by the evaluation method according to the embodiment of the present invention (FIG. 15). Regarding activity, when placebo was administered to Alzheimer's disease model mice and normal littermate mice, the activity of the attractant zone was high in the former (Fig. 15, upper left figure), and the activity of the relief zone was high in the latter (Fig. 15). Upper right figure). In contrast, in Alzheimer's disease model mice treated with Compound K, activity increased in the zone between the attractant zone and the relief zone (upper center figure in Fig. 15), and the behavioral abnormalities characteristic of dementia were normal. There were signs of change in behavior. Regarding Trajectory, the degree of accumulation of Trajectory in Alzheimer's disease model mice treated with Compound K (lower center of FIG. 15) is between the degree of accumulation in Alzheimer's disease model mice treated with placebo and the degree of accumulation in normal littermate mice. It showed a typical feature.

We also performed histopathological analysis of the brains of Alzheimer's disease model mice treated with placebo, normal littermate mice, and Alzheimer's disease model mice treated with compound K. As a result, in the brains of Alzheimer's disease model mice administered with Compound K, the formation sites of geriatric plaques (upper center figure in FIG. 16) and changes in neurofibrils (lower center figure in FIG. 16) decreased, and normal littermate mice administered with placebo. (Fig. 16, upper right figure and lower right figure), the therapeutic effect of compound K could be confirmed.
From the above results, it was clarified that the method for evaluating an experimental animal according to an embodiment of the present invention can be used for screening a therapeutic agent for a disease associated with spontaneous dyskinesia.

The present invention provides a non-invasive, non-learning, self-issued behavioral disorder evaluation method for laboratory animals and equipment for carrying out the method. Therefore, by using the equipment and method of the present invention, behavioral abnormalities of experimental animals can be evaluated easily and quickly, and by using the method of the present invention, screening of compounds effective for behavioral abnormalities can be easily performed. be able to.
The present invention is highly expected to be used in the medical field.

Code 10; Compartment code 11; Attraction installation position code 12: Behavior restriction tool installation position code 20: Detection means code 21; Video means code 22; Control means code 23; Signal line

Claims (11)

A facility for evaluating the presence or absence of abnormalities in the self-issued movement of a laboratory animal, which is provided with a compartment for accommodating the laboratory animal, and an attractant and a behavior restriction device are installed in the compartment. The shortest distance between the partition of the compartment and the outer surface of the behavior-restricting device , which attracts the laboratory animal and is an obstacle to the behavior of the behavior-restricting device. However, the equipment in which the behavior restriction tool is installed so as to be about 1.0 to about 1.5 times the width of the body of the experimental animal . The distance from the outer surface of the behavior restriction device to the outer surface of the attractant so that the shortest distance is about 1.5 to about 2.0 times the width of the body of the experimental animal. The equipment according to claim 1 in which an attractant is installed. A detection means for detecting an experimental animal in the compartment, a video means for photographing the inside of the compartment, and a control means for operating the video means and acquiring an image captured by the video means as a video signal. The equipment according to claim 1 or 2 , further comprising a detection means including. A method for evaluating the presence or absence of abnormalities in the self-issued behavior of an experimental animal using the equipment according to any one of claims 1 to 3 , which is obtained by analyzing an image of the experimental animal acquired by the control means. An evaluation method using the activity and / or trajectory of the experimental animal as an index. A method for evaluating the presence or absence of cognitive dysfunction in an experimental animal by using the method for evaluating the presence or absence of abnormal self-issued behavior of the experimental animal according to claim 4 . The evaluation method according to claim 5 , which includes the following steps (a) to (c).
(A) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal of the control,
(B) The step of measuring the activity in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the attractant obtained in the attractant zone obtained in step (b). The experimental animal has cognitive function when it is significantly higher than the activity in the zone and / or when the activity in the safe zone obtained in step (b) is significantly lower than the activity in the safe zone obtained in step (a). Process to evaluate as having a disability The evaluation method according to claim 5 , which includes the following steps (a) to (c).
(A) A step of determining the degree of accumulation of Trajectory in the attractant zone and / or the safe zone of the experimental animal of the control.
(B) The step of determining the degree of accumulation of Trajectory in the attractant zone and / or the safe zone of the experimental animal to be evaluated, and (c) the degree of accumulation of Trajectory in the attractant zone obtained in step (b) is the step (a). ) Is higher than the degree of accumulation of Trajectory in the attractant zone and / or the degree of accumulation of Trajectory in the safe zone obtained in step (b) is higher than the degree of accumulation of Trajectory in the safe zone obtained in step (a). Step to evaluate that the experimental animal has cognitive impairment when it is low The disease exhibiting the cognitive dysfunction is any one of Alzheimer's dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disorder and intellectual disability based on the aftereffects of the disease. The evaluation method according to any one of claims 5 to 7 , wherein the evaluation method is characterized by the above. The method according to any one of claims 4 to 8 , wherein the experimental animal is a rodent or a small primate. A method for screening a compound effective for treating a disease associated with spontaneous behavioral disorder by using the method according to any one of claims 4 to 9 . The disease showing the self-issued dyskinesia is one of Alzheimer's disease, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disability and intellectual disability based on the aftereffects of the disease. The method according to claim 10 , wherein the method is characterized by the above.

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