JP2018143112A - Facility and method for evaluating animal's behavior - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a non-learning type method for evaluating a spontaneous behavioral abnormality on an experimental animal, in which no stress is applied (non-invasive), and a facility for executing the method.SOLUTION: There is provided: a facility for evaluating a behavior of an experimental animal including a compartment 10 for housing an experimental animal, in which an attractant and a behavior limiting tool are installed in the compartment 10; and a method for evaluating presence or absence of a spontaneous behavioral abnormality on the experimental animal, using the facility. The evaluation method uses as indexes the activity and/or trajectory of the experimental animal acquired by analyzing a video of the experimental animal acquired by control means.SELECTED DRAWING: Figure 1

Description

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

  Alzheimer's disease is a symbol of a neurodegenerative disease whose main symptom is a decline in memory ability, which is mainly caused by degeneration and disappearance of hippocampal neurons that control memory. Neuropathologically, the neurodegenerative region of Alzheimer's disease extends beyond the hippocampus to the entorhinal cortex (parahippocampal gyrus), hippocampal limbs, Nucleus basalis of Meynert, and amygdala. Therefore, Papez memory circuit (hippocampus → cerebral arch → papillary body → papillary thalamic tract: Vicq d'Azyr bundle → prethalamic nucleus → zonal gyrus → zonal bundle → hippocampus Closed circuit) and Yakovlev emotional circuit (amygdala → dorsalis [MD]) → prefrontal cortex (prefrontal orbital 1/4 cortex region) → peritonsillar cortex → amygdala closing circuit ) Is also a disorder that causes emotional disorder and reduced thinking ability in addition to memorizing ability, and overall cognitive function is reduced (Non-patent Document 1). For this reason, Alzheimer's disease is called dementia.

  The number of Alzheimer's disease patients has increased rapidly in Japan in recent years, and drought, disappearance, traffic accidents, etc. caused by this disease are becoming social problems. There are approximately 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 daily environment requires care support for caring for the patient. For this reason, the current situation is that not only the patient himself but also the patient family and the caregiver are forced to bear a great burden physically, mentally and economically. In 2045, it is estimated that 1 in 2 people over the age of 65 will have Alzheimer's disease. In other words, Alzheimer's disease is the most costly monetary disease in developed countries including Japan from the viewpoint of economic cost-effectiveness ratio, and it is said to be a national disease. Nevertheless, no effective drug has been established for the treatment of Alzheimer's disease.

As for the diagnosis of Alzheimer's disease, a so-called noninvasive diagnostic method has been established. Here, “non-invasive” means that when the patient is diagnosed as having Alzheimer's disease, a biopsy histological diagnosis from the brain when making a definite diagnosis of a brain tumor patient is not performed. That is, it does not implement the physical invasive stimulus evaluation method in which a specific intellectual task is given to a patient to learn it, and if it is not learned well, a thermal stimulus or an electrical stimulus is given. Specifically, the Wechsler Adult Intelligence Scale (WAIS), which accurately evaluates the higher-order functions that Alzheimer's disease patients have at the time of testing, and the Hasegawa dementia simplified intelligence evaluation scale (Hasegawa dementia) Two higher-order function evaluation methods (rating scale-revised: HDR-R) have been established. In the Wexler intelligence test, judgment is made mainly based on evaluation based on two elements, namely, intelligence quotient (IQ) and behavioral intelligence. The Hasegawa simplified intelligence evaluation scale mainly evaluates with linguistic intelligence. When Alzheimer's disease is diagnosed by these clinical evaluation systems, this is generally called clinical Alzheimer's disease.
An accurate final diagnosis of Alzheimer's disease is made with histopathological evaluation. That is, there is no final diagnosis of Alzheimer's disease without histopathological evaluation. In other words, a definitive diagnosis of Alzheimer's disease is made with a histopathological diagnosis of the post-mortem brain of a clinical Alzheimer's disease patient.

  On the other hand, there is no naturally occurring highly cognitively impaired mouse corresponding to human Alzheimer's disease in the natural world. Therefore, the evaluation of the cognitive function of the mouse is basically based on the premise that the mouse can learn. For this reason, conventional mouse cognitive function evaluation methods do not assume mice with advanced cognitive impairment such as human Alzheimer's disease, and are not noninvasive methods. 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) The evaluation method such as 2) is a method for evaluating the degree of learning of a mouse by applying invasive stress to the mouse individual. Normally, such an evaluation method requires a large number of trials in order to ensure the accuracy of the evaluation. Therefore, in the evaluation of the cognitive function of a mouse, the risk of dying of the mouse increases due to stress on the mouse individual.

At present, as a model mouse for human Alzheimer's disease, a double transgenic mouse in which a human amyloid precursor protein gene and a human tau protein gene, and respective human mutant genes are introduced into the mouse is histopathologically closest to a human Alzheimer patient. . The cost of this double transgenic mouse is very expensive, for example, about 10 million yen in the original line. In an experiment using such an expensive Alzheimer's disease model mouse, in order to make an accurate final diagnosis of the result, the model is usually killed at regular intervals and histopathological evaluation of the brain is performed. Therefore, the experiment using the Alzheimer's disease model mouse can be said to be an extremely expensive experimental system.
Although it is assumed that the model mouse that 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 suitable for the two points of adding a lot of stress that leads to fatal accidents. In addition, a histopathological evaluation method for obtaining a definitive diagnosis at regular intervals is not suitable from the viewpoint 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 perform cognitive function evaluation using a model mouse simply, 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 self-issued behavior of laboratory animals, and is a non-learning and non-stressing (non-invasive) method based only on the daily behavior of laboratory animals. I will provide a.
Moreover, this invention provides the installation used for the said evaluation method.
Furthermore, the present invention provides a screening method for compounds that affect the self-issue behavior of experimental animals.

The inventors have provided a space for allowing the experimental animal to pass between the cage wall and the cage wall (about the width of the body of the experimental animal), and a tool (a tool that restricts the behavior of the mouse, hereinafter). "Behavior restriction tool") and a facility in which food and water (hereinafter "attractant") were installed at a position about 1.5 times the body width of the experimental animal from the behavior restriction tool.
The inventors used mice (Alzheimer's disease model mice and normal littermate mice) as animals for observing behavior (spontaneous behavior), and recorded only spontaneous daily behavior of mice for 10 minutes and analyzed the behavior. It was. In conducting the behavior analysis, Activity and Trajectory were used as behavior evaluation elements.
In the above-mentioned facilities developed by the inventors, the area between the area where the attractant can be easily accessed (the place where food and water are located and its vicinity; the “attractant zone”), the action restriction device and the breeding cage Space ("comfortable zone") is formed.

When the behavior analysis of mice was performed in a cage without a behavior restriction device, there was 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 with restriction devices, the activity of the Alzheimer's disease model mice group was the attractant zone: 48.37 ± 5.24, the safety zone: 17.09 ± 5.45, and the normal litter mouse group was the attractant. The zone was 8.95 ± 8.60, and the safety zone was 56.53 ± 17.35.
That is, Alzheimer's disease model mice had significantly higher activity in the attractant zone, and abnormal feeding behavior and decreased alertness were observed. Spatial cognitive impairment that activity in the safe zone that is assumed to feel secure is significantly lower in Alzheimer's disease model mice than in normal littermate mice, with reduced risk avoidance ability and low activity in the space where they should be placed Admitted.
As described above, it has been clarified that non-learning type non-stress type experimental animal behavior evaluation can be performed by using the equipment developed by the inventors.

The present invention has been completed based on the above findings.
Therefore, the present invention includes the following (1) to (12).
(1) A facility for evaluating the behavior of a laboratory animal, comprising a compartment for accommodating a laboratory animal, in which an attractant and a behavior restriction device are installed.
(2) The distance between the compartment partition and the outer surface of the action restriction device, the shortest distance being about 1.0 to about 1.5 times the width of the body of the experimental animal, The facility according to (1), wherein the action restriction tool is installed.
(3) The distance from the outer surface of the action restriction device to the outer surface of the attractant, the shortest distance being about 1.5 times to about 2.0 times the width of the body width of the experimental animal The equipment according to (1) or (2) above, wherein the attractant is installed.
(4) Detection means for detecting an experimental animal in the compartment, wherein the video means for photographing the inside of the compartment and the video means are operated, and the video photographed by the video means is acquired as a video signal. The facility according to any one of (1) to (3), further including detection means including control means.
(5) A method for evaluating the presence or absence of an abnormality in the self-issued behavior of a laboratory animal using the equipment according to any one of (1) to (4) above, wherein a video of the laboratory animal acquired by the control means is displayed. An evaluation method using the activity (activity) and / or Trajectory (center of gravity trajectory) of the experimental animal as an index obtained by analysis.
(6) A method for evaluating the presence or absence of cognitive dysfunction in an experimental animal using the method for evaluating the presence or absence of an abnormality in self-issued behavior of the experimental animal according to (5) above.
(7) The evaluation method according to (6), including the following steps (a) to (c):
(A) measuring activity in an attractant zone and / or a relief zone of a control laboratory animal;
(B) a step of measuring the activity in the attract zone and / or the relief zone of the experimental animal to be evaluated, and (c) the attract in which the activity in the attract zone obtained in step (b) is obtained in step (a). If the activity is significantly higher than the activity in the zone and / or 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) for evaluating that there is a failure. The evaluation method according to (6), comprising the following steps (a) to (c).
(A) determining the accumulation of Trajectory in the attractant zone and / or the relief zone of the control laboratory animal,
(B) determining the accumulation degree of the Trajectory in the attractant zone and / or the relief zone of the experimental animal to be evaluated; and (c) determining the accumulation degree of the Trajectory in the attractant zone obtained in the step (b) ) Is higher than the degree of trajectory accumulation in the attractant zone obtained in step (b) and / or the degree of trajectory accumulation in the relief zone obtained in step (b) is greater than the degree of trajectory accumulation in the relief zone obtained in step (a). (9) The disease showing the cognitive dysfunction is Alzheimer-type dementia, cerebrovascular dementia, Lewy body dementia, manic depression The evaluation method according to any one of (6) to (8) above, wherein the evaluation method is any one of intellectual impairment based on schizophrenia, developmental disorder, and sequelae 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 accompanied by a self-issued dysfunction using the method according to any one of (5) to (10) above.
(12) The disease showing the self-issued dysfunction is any of Alzheimer's dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disorder and intellectual disability based on the sequelae of the disease. The method according to (11) above, wherein

  According to the facility and the evaluation method of the present invention, it is possible to perform the self-issue of a laboratory animal simply, rapidly, and at low cost by a non-invasive and non-learning method.

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

The schematic of the installation concerning this invention. Reference numeral 10: compartment, reference numeral 11: attractant installation position, reference numeral 12: action restriction tool installation position, reference numeral 20: detection means, reference numeral 21; video means, reference numeral 22: control means, reference numeral 23; signal line The equipment concerning the present invention used in the example. A video camera for photographing the behavior of the experimental animal from above and a general-purpose computer for operating the video camera and acquiring images are connected. The photo was taken under bright field conditions. The layout of the food and water (attractant) box and the toy for mice (behavior restriction tool) in the compartment of the equipment used in the examples. A is a layout in an ordinary breeding cage (compartment) where only a food and water box is installed without installing a toy mouse (behavior restriction device) (Open field condition; food and water in the usual breeding cage (attract)) This is the condition where only B is a layout diagram in a normal breeding cage in which a mouse toy (behavior restriction tool) is placed in an open field (With Toy condition; a condition in which a mouse toy (behavior restriction tool) is placed in an open field condition). Illustration of the cage bottom divided into 5 parts according to the spatial differences that occur under With Toy conditions for quantitative quantification of Activity. Statistical analysis was performed by arbitrarily dividing the analysis field using Smart 3.0 used in this analysis and comparing the values for each division. Image excerpted from behavior analysis video of normal littermate mice under open field conditions. Mice (A) that were diagonally away from food and water moved towards food and water (B) and further moved towards food and water (C). After moving, the behavior which showed interest in food and water was performed (D). In addition, an irregular behavior pattern such as returning to the position (A), not just near food and water, was shown. Results of behavioral analysis 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 breeding cage (hereinafter the same in FIGS. 6A, 8A, 10A and 12A). Activity activity is high in two places around the bait and water and in the vicinity of the normal rearing cage angle away from the location. B is a view of the trajectory trace of a mouse moving in a normal breeding cage as seen from above (hereinafter the same in FIGS. 6B, 8B, 10B, and 12B). Trajectory corresponds to Activity, It accumulated near the food and water, and around the upper right corner of the normal rearing cage. 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 the food and water box (C). Similar to normal littermate mice, after moving, behavior that showed interest in food and water was performed (D). An irregular behavior pattern such as returning to the position (A) as in the normal littermate mouse was shown. Results of behavioral analysis 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 in the vicinity of the normal rearing cage angle away from that position (A). In addition, Trajectory was accumulated in the vicinity of food and water, and in the vicinity of the upper right corner in the normal breeding cage apart from the food, as in normal littermate mice (B). An image excerpted from a behavior analysis video of a normal littermate mouse with With Toy conditions. The experiment was started from the position between the position of the food and water and the position of the mouse toy. Although food and water were confirmed (A), it did not move in that direction, but moved to Zone 4 (safe zone) surrounded by toys for mice and a normal cage wall (B). After that, he stayed in Zone 4 (Reliable Zone) for a long time and was active in the place (C and D). Behavior analysis results of normal littermate mice with With Toy conditions. Image of Activity 3D graph (A) and Trajectory (B). Activity in Zone 4 (reliable zone) was very high, and decreased as it approached the vicinity of food and water (A). In addition, Trajectory was abundantly accumulated in the position where Activity was high (B). An image excerpted from a behavior analysis video of a model mouse with Alzheimer's disease under With Toy conditions. The experiment was started from the same position as normal littermate mice. Alzheimer's disease model mice confirmed their food and water (A), moved in that direction (B), and took actions with strong interest, such as eating and smelling. After that, it was confirmed that it appeared frequently in the vicinity of the installation position of food and water (C and D). Results of behavioral analysis of Alzheimer's disease model mice with With Toy conditions. Activity 3D Graph (A) and Trajectory (B) images. Activity was very high near food and water and low in Zone 4 (safe zone), which was high in normal littermate mice (A). Trajectory was accumulated in a circle around the toy for the mouse, and accumulation in the vicinity of the normal cage wall (safe zone) was not observed (B). The graph which shows the ratio (Activity%) in each Zone of Activity in With Toy conditions. The activity ratio (Activity%) of Alzheimer's disease model mice (black graph: 3 mice) and normal littermate mice (white graph: 4 mice) in each of the 5 zones was graphed. When Mann-Whitney U test was performed, there was a significant difference of * p <0.05 between Zone2 and Zone4. The correspondence between the activity% (FIG. 13) and Trajectory (FIG. 12B) results of Alzheimer's disease model mice and the histopathological analysis results is shown. Effect of Compound K that can suppress senile plaque formation and neurofibrillary tangles. When Compound K was administered to an Alzheimer's disease model mouse, it was examined what changes occurred in Activity (upper) and Trajectory (lower) of the mouse. The activity pattern of the Alzheimer's disease model mice to which Compound K was administered (upper row) showed an intermediate pattern between the image patterns of the placebo-treated Alzheimer's disease model mice and the placebo-treated normal littermate mice. The results of histopathological analysis of the brain of Alzheimer's disease model mice administered with Compound K are shown. We compared the formation of senile plaques and neurofibrillary tangles in the brains of placebo-treated Alzheimer's disease mice and normal littermate mice, and Compound K-administered Alzheimer's disease model mice. Alzheimer's disease mice treated with placebo have many senile plaques and neurofibrillary tangles. Both structures are not observed in normal littermate mice treated with placebo. In Alzheimer's disease model mice administered with Compound K, the formation of senile plaques and neurofibrillary tangles is markedly suppressed compared to placebo-administered Alzheimer's disease model mice.

A first embodiment of the present invention is a facility for evaluating the behavior of a laboratory animal, comprising a compartment for accommodating a laboratory animal, and an attractant and a behavior restriction device are installed in the compartment. Equipment.
The equipment according to the first embodiment includes a compartment (reference numeral 10 in FIG. 1) that can accommodate experimental animals. Here, the “compartment” refers to a space in which a laboratory animal can be accommodated (into) and is 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 raising animals. The material of the compartment partition may be any material as long as it is a material that is not easily destroyed by the housed laboratory animal, and may be, for example, metal, plastic, glass, wood, or the like. Further, the partition may have any shape such as a net shape or a plate shape. The shape of the compartment (partitioned space and the shape of the space seen from above the equipment) is not limited, for example, it may be a rectangular shape common to cages of domestic animals, Alternatively, the shape may be any shape such as a circle, an ellipse, a triangle, and a diamond.
The size of the compartment (bottom area enclosed by the partition) is limited so long as the experimental animal can freely move and is large enough to perceive the attractant and the action restriction device during the action. Not. For example, when a mouse is used as an experimental animal 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 40cm to about 70cm. The height of the compartment is sufficient if the experimental animal cannot escape, for example, when using a mouse, 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 likes, or an object that affects the instinct of the experimental animal and attracts the animal. Thus, for example, in addition to food and water, it may be an addictive drug, a pheromone that attracts experimental animals to be used, and the like. As for the attractant, only the attractant may be placed in the compartment, but if necessary, it may be placed in an appropriate container and placed in the compartment.
In addition, the “behavior restriction tool” (reference numeral 12 in FIG. 1) installed in the compartment of the first embodiment is an object that hinders the behavior of experimental animals. For example, it is not a thing that the experimental animal can easily get over, but a thing that restricts its behavior, such as changing the traveling direction of the experimental animal, is preferable. The action restriction device 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 (screen shape).
The size (volume) of the action restriction device is desirably larger than the volume of the body of the experimental animal to be used. For example, the volume of the body of the experimental animal to be used is about 1 to about 2,500 times, more preferably about 10 times to About 1,000 times. For example, when a mouse is used as an experimental animal, the size of the restriction tool is preferably about 10 to 1,000 times the mouse volume.

It is desirable that the behavior restricting device is installed in the compartment with an interval between the compartment partition and the behavior restricting device so that the experimental animal can be placed. For example, it is preferable that the distance between the compartment partition and the outer surface of the action restriction device is about 1.0 to about 1.5 times the length of the width of the body of the experimental animal to be used.
When a mouse is used as an experimental animal, it is preferable that the shortest distance is, for example, about 2 cm between the compartment partition and the outer surface of the action restriction device.
In the present specification, a space area formed between the compartment partition and the action restriction tool is referred to as a “safe zone”.

The position of the attractant in the compartment is preferably a position where the experimental animal cannot reach the attractant unless the experimental animal moves from the “reliable zone”, and a position where the experimental animal can perceive the attractant from the “reliable zone” is preferable. . 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 times to about 3.0 times the lateral length of the body of the experimental animal to be used, about 1.5 About twice 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. When the attractant is placed in a container or the like, the outer surface including the container is included. It is the surface.
When a mouse is used as the experimental animal, it is preferable that the shortest distance is, for example, about 4 to 80 cm, which is the distance from the outer surface of the action restriction device to the outer surface of the attractant.
In the present specification, a range in which an experimental animal can contact an attractant without moving is referred to as an “attractant zone”.

  The facility according to the first embodiment may further include detection means 20 for detecting the behavior of the experimental animal in the compartment (FIG. 1). The detection means 10 includes a video means 21 such as a video camera for photographing a laboratory animal, a control means 23 such as a computer for operating the video means to acquire an image taken by the video means as a video signal, and a video. It comprises a signal line 22 for transmitting and receiving signals between the means and the control means. Here, the computer as the control means receives the video signal of the experimental animal and displays not only the continuous video obtained by photographing the experimental animal on the computer display but also software for analyzing the continuous video. It may be.

The second embodiment of the present invention is a method for evaluating the presence / absence of abnormality in the self-issued movement of a laboratory animal using the equipment according to the first embodiment, and is a control means provided in the first embodiment. Is an evaluation method using the 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 the method.
Self-issued movement is a general term for actions related to life support based on instinct, such as movement, climbing, tail chasing, standing up, grooming, and feeding behavior.
Here, Activity and Trajectory can be obtained by analyzing a video of an experimental animal. The time for photographing the experimental animal varies depending on the experimental animal to be used, and may be any time necessary 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 1 hour, more preferably about 10 and several minutes. .
Activity is the sum of the self-issued amount of movement. For example, the amount of fluctuation of a specific body surface point of the experimental animal (for example, the center of gravity of the experimental animal, the body surface such as the head, tail, etc.) relative to the compartment (for example, It can be quantified as the movement speed or movement distance of a specific body surface point. Activity quantification is based on analysis software (eg Smart 3.0, Panlab SLU, Barcelona, Spain; PhenoScan series, Prime Tech, Tokyo, Japan; EthoVision XT, Nordus Information Technology, Wageningen) , Netherlands; CompACT series, Muromachi Kikai Co., Ltd., Tokyo, Japan; ANY-maze, Stoelting Co., Wood Dale, Illinois, USA; ABDigitizer, Intelligent Information Systems, Kyoto, Japan, etc.) Can be easily implemented.

  Trajectory tracks experimental animals that perform self-issued movements, and traces them as lines. In a region where many self-issued movements of experimental animals are observed, lines depicting Trajectory accumulate (the number of trajectory lines increases). The degree of Trajectory accumulation in a certain area can be determined, for example, by counting the number of lines drawn on the locus of the center of gravity of the experimental animal in that area. For example, FIG. 10B shows a track of the center of gravity in the compartment as a line by tracking the experimental animal and using analysis software. Since the number of trajectory lines in the secure 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 secure zone is higher than in the other zones. Trajectory is an analysis software based on information tracked on experimental animals (eg Smart 3.0, Panlab SLU, Barcelona, Spain; PhenoScan series, Prime Tech, Tokyo, Japan; EthoVision XT, Nordus Information Technology, Wageningen, Netherlands) ; CompACT series, Muromachi Kikai Co., Ltd., Tokyo, Japan; ANY-maze, Stoelting Co., Wood Dale, Illinois, USA; ABDigitizer, Intelligent Information Systems Co., Ltd., Kyoto, Japan; Imagepower TrackingEye, Fujitsu Kyushu System Service Co., Ltd. , Fukuoka, Japan, etc.) can be easily drawn. By counting the number of trajectory lines at an arbitrary place in the compartment or by estimating the degree of trajectory line accumulation by visual observation, the degree of Trajectory accumulation at the arbitrary place can be determined.

  The evaluation method according to the second embodiment of the present invention compares the activity and / or trajectory of an experimental animal to be evaluated with the activity and / or trajectory of a control experimental animal (experimental animal whose normal behavior is normal). Thus, when a result different from that of a control experimental animal is obtained, it is evaluated (or determined) that the experimental animal to be evaluated has some spontaneous issue abnormality. When comparing activities, activities can be easily compared by quantifying them as described above. As for Trajectory, it is possible to evaluate and compare the degree of accumulation in a specific region within the compartment of the trajectory (trajectory of the center of gravity of the animal) of each experimental animal (control and evaluation target animal).

The abnormality of the self-issued behavior is also observed in cases of developing cognitive dysfunction (for example, dementia, intellectual depression based on manic depression, schizophrenia, developmental disorder and sequelae of illness).
Therefore, the third embodiment of the present invention is a method for evaluating the presence or absence of cognitive dysfunction in a laboratory animal using the method for evaluating the presence or absence of abnormality in the self-issued behavior of the laboratory animal according to the second embodiment. It is. When a self-issue dysfunction characteristic of cognitive impairment is recognized in the animal to be evaluated using the method for evaluating the self-issue motility abnormality of the experimental animal according to the second embodiment, the test animal to be evaluated has a cognitive function. It can be evaluated (or judged) if there is a suspicion of a disorder.
When Activity is used as an evaluation index of self-issued movement abnormality, the presence or absence of cognitive dysfunction in experimental animals can be evaluated (determined) by performing the following steps (a) to (c).
(A) measuring activity in an attractant zone and / or a relief zone of a control laboratory animal;
(B) a step of measuring the activity in the attract zone and / or the relief zone of the experimental animal to be evaluated, and (c) the attract in which the activity in the attract zone obtained in step (b) is obtained in step (a). The experimental animal has cognitive impairment when significantly higher than the activity in the zone and / or 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 that there is

When Trajectory is used as an evaluation index of self-issued movement abnormality, the presence or absence of cognitive dysfunction in experimental animals can be evaluated (determined) by performing the following steps (a) to (c).
(A) determining the accumulation of Trajectory in the attractant zone and / or the relief zone of the control laboratory animal,
(B) determining the accumulation degree of the Trajectory in the attractant zone and / or the relief zone of the experimental animal to be evaluated; and (c) determining the accumulation degree of the Trajectory in the attractant zone obtained in the step (b) ) Is higher than the degree of trajectory accumulation in the attractant zone obtained in step (b) and / or the degree of trajectory accumulation in the relief zone obtained in step (b) is greater than the degree of trajectory accumulation in the relief zone obtained in step (a). In the case where the experimental animal has a low cognitive function, the evaluation of the presence or absence of cognitive dysfunction in the experimental animal may be performed using both Activity and Trajectory as indicators.

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

The evaluation methods according to the second and third embodiments evaluate the presence / absence of abnormality in self-issued movement and the presence / absence of cognitive dysfunction. Therefore, it can be used for screening compounds effective for the treatment and / or prevention of diseases exhibiting abnormal self-issued behavior, for example, diseases associated with cognitive impairment.
A candidate compound for treatment (or prevention) of the disease is administered to a disease model animal exhibiting self-issue dysfunction evaluated by the evaluation method according to the second and third embodiments, and the self-issue dysfunction is improved, The candidate compound is used in the case where it becomes self-issued in the same manner as a normal experimental animal, or when it shows intermediate behavior between the self-issued behavior of a disease model animal and the normal experimental animal. Therefore, it can be determined that it is effective for the treatment of the disease.
In particular, when a model animal having a pathological characteristic characteristic of a disease such as an Alzheimer type dementia model mouse (for example, Tg (APPSWE) 2576Kha Tg (Prnp-MAPT * P301L) JNPL3HImc mouse, etc.) is used, Correspondence (association) between abnormalities of self-issued movement evaluated by the embodiment of the invention and pathological characteristics can be performed (see the examples), and only by the evaluation method of the present invention (pathological diagnosis) Simple screening of candidate compounds is possible.

  The experimental animals used in the embodiments of the present invention are not particularly limited. For example, in addition to small animals such as rodents (for example, mice, rats, hamsters, guinea pigs, and pineapples), small primates (common marmosets) Etc.) are preferred.

The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, throughout this specification, where the word “a”, “an”, and “the” is included, the term “a”, “an”, and “the” includes plurals as well as the singular unless the context clearly indicates otherwise. Including things.
The present invention will be further described below with reference to examples. However, the examples are merely examples of the embodiments of the present invention, and do not limit the scope of the present invention.

Materials and Methods Mouse cage (compartment) used for analysis system (equipment according to the present invention)
Ordinary mouse breeding cage (Part No .: KE-3101, Theobit Corporation, Tokyo, Japan) and 2 mouse feeds (Product Name: CE-2, Nippon Clare Corporation, Tokyo, Japan) : 3.5g) and drinking water for mice (water from the tap water, which is human drinking water) filtered using a filter with a filtration accuracy of 0.5μm (manufacturer: Aion, trade name: KANEFIEL, model: FD-005) Use) A box made of paper of 113mm x 81mm x 15mm (height) with a glass container (trade name: BT-500 lid, Matsunami Glass Industry Co., Ltd., Wada, Osaka, Japan) poured into it (about 5ml) A bait / water box (attractant) was placed, and a mouse toy (trade name: K3570 / Mouse Igloo (Blue), Bio Serv, Flemington, New Jersey, USA) (behavior restriction device) was also placed. This cage is covered with a flooring (trade name: Pure Chip, Shimizu Experimental Materials Co., Ltd., Kyoto, Japan) about 1 cm thick, and a food and water box and a toy for a mouse were placed on this.

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

3. Peripheral device used for analysis system General-purpose personal computer (Model: ASPIRE V3-571-H54D / K, manufacturer: Acer Incorporated, New Taipei, Republic of China), [OS: Windows 7 Home Premium, manufacturer: Microsoft Corporation , Raymond, Washington, USA], the camera data capture software (trade name: Microsoft Visual Studio, manufacturer: Microsoft Corporation, Raymond, Washington, USA) was modified to prepare the shooting environment. In this system, the camera is automatically activated by an operation from a personal computer (personal computer) and can shoot for 10 minutes. The captured video was analyzed using analysis software (trade name: Smart 3.0, Panlab SLU, Barcelona, Spain). The moving image recording in this example was 10 minutes after the mouse was placed, and the total recording capacity was set to be about 20 MB per recording. All of this data was set so that it could be automatically recorded on a personal computer (Fig. 2). Peripheral devices used in the analysis system were installed as shown in FIG.
Place the mouse cage for analysis so that the short side of the normal mouse cage and the long side of the food and water box overlap 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 cage. It installed so that it might be between. Moreover, although the water glass container was installed in 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 opposite to the position where the water glass container was placed (FIGS. 3 and 4).

4). Zone setting in a normal breeding cage (With Toy) with a mouse toy
In the normal breeding cage under the With Toy condition, five zones were set as shown in FIG. Zone2: Zone where mice can eat food (Bait Zone; Attract Zone), Zone3: Zone where toys are placed, Zone4: Zone that seems to be a burrow in nature (Comfortable Zone: Comfortable and safe for mice) Zone, which is supposed to give the zone (safe zone), and Zone5, which is considered to be the calmest in normal mice, is not close enough to eat, but is interested in food and water Zone that seems to be, Zone1: Zones that are the above-mentioned Zone2 to Zone5 subtracted from the total bottom (Total), respectively. In this example, the setting (= Detection Settings) was optimized according to the color difference between the color of the mouse hair and the color of the toy for the mouse. 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. Experimental animals As strains of Alzheimer's disease mice, three strains: Tg (APPSWE) 2576KhaTg (Pmp-MAPT * P301L) JNPL3HImc mice (Taconic, New York, USA) were used, and normal mice were littermate mice ( 4 Taconic, New York, USA) were used.
The Tg (APPSWE) 2576KhaTg (Pmp-MAPT * P301L) JNPL3HImc mouse is a human mutated APP gene (Sweden) in which the 670th lysine of the human amyloid precursor protein (APP) gene is mutated to methionine and the 671th asparagine is leucine. This is a double-transgenic mouse produced by introducing a mutated human tau mutant gene in which the 301th proline of human tau protein gene is point-mutated to leucine. This Alzheimer's disease model mouse expresses senile plaques and neurofibrils at approximately 1 year of age.

6). Statistical analysis method Quantitative numerical values of Alzheimer's disease model mice and normal littermate mice between each of Zone 1 to Zone 5 when a toy for a mouse was installed were expressed as mean ± standard deviation. All statistical analyzes were performed using Macintosh Software StatView: Version 5.0 (SAS Inc., Cary, NC, USA). For the significance test, the Mann-Whitny U test was used, and it was assumed that there was a statistically significant difference because the risk factor was less than 0.05.

7). Histopathological analysis Compound K and placebo (solvent of Compound K) capable of suppressing the formation of senile plaques and neurofibrils were administered to experimental animals. As for the oral administration method, the volume was accurately measured with a plastic syringe, and a gastric sonde for mice was directly connected to the plastic syringe, which was reliably administered orally and transesophageally.
As experimental animals, human Alzheimer's disease type dementia model mice (for example, a gene having a Swedish mutation of a gene encoding 695 amino acids of amyloid β (Aβ) precursor protein and a 301 th proline of a human tau protein gene) Characteristic for diseases such as Tg (APPSWE) 2576KhaTg (Prnp-MAPT * P301L) JNPL3HImc strain Alzheimer's disease model double transgenic mice, which highly express both human genes of the mutant gene mutated to leucine (P301L) Model animals with different histopathological features are used. Similarly, normal littermate mice were also used.

Identification of senile plaques and neurofibrils appearing in 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, the core protein of senile plaques, and phosphorylated tau protein, 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. For detection of Aβ40, an anti-amyloid β protein (1-40) mouse monoclonal antibody (clone No. BA27) in the kit was used. For detection of Aβ42, an anti-amyloid β-protein (1-42) mouse monoclonal antibody (clone No. BC05) in the kit was used. Finally, it was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Dako, Glostrup, Denmark) as a color former. As a method for detecting phosphorylated tau protein, a combination of the following primary antibody and ABC (avidin-biotin-immunoperoxidase complex) method was performed. As the primary antibody, an anti-phosphorylated tau protein (PHF-tau) mouse monoclonal antibody (clone: AT8, Innogenetics: currently under the name Fujirebio Inc., Tokyo) was used. The ABC kit used was 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 stained samples of HE staining, Aβ40 immunostaining, Aβ42 immunostaining, and AT8 immunostaining (BX41: Olympus) was examined and image analysis was performed along with taking pictures with the device.
The above experiment was conducted with the approval of the Tottori University Animal Experiment Committee.

Result 1. Results when only food and water boxes are installed in the rearing cage (compartment) (Open field conditions) Analysis under the Open field conditions where no toys for mice (behavior restriction devices) are installed Short side: 81mm x height: 15mm box containing about 5ml of drinking water for mice and 2 pieces of 3.5g of food (food / water box) (attractant) The analysis was carried out in a state where it was placed in a corner.

1-1. Results of behavioral analysis of normal littermate mice under open field conditions.
Regarding the behavior of normal littermate mice, the experiment was started from a position opposite to the position where food and water were placed (FIG. 5A). First, we approached the vicinity of food and water, which are strong instincts (FIG. 5B). And when it reached the vicinity of food and water, it sniffed the food and showed interest (FIGS. 5C and D). However, this state does not continue, it leaves in a short time and moves in the direction opposite to the installation position of food and water, or reverses and moves again in the vicinity of food and water, It did not show a certain behavior inclination pattern.
FIG. 6 shows the activity and trajectory (center of gravity locus) in a 10-minute analysis of normal littermate mice under open field conditions. Activity was high at two places around the bait and in the vicinity of the upper right corner in the opposite direction away from it (FIG. 6A). In addition, Trajectory was accumulated around the upper right corner of the cage on the opposite side of the position where the food and water were installed so as to correspond to Activity (FIG. 6B).

1-2. Results of behavioral analysis of Alzheimer's disease model mice under open field conditions.
The image showing the behavior of the Alzheimer's disease model mouse was started from the position opposite to the position where the food and water were placed, as in the case of the normal littermate mouse for easy comparison (FIG. 7A). Alzheimer's disease model mice approached food and water, which are instinctive stimuli (FIG. 7B), and showed interest in sniffing food and water (FIGS. 7C and D). However, in a relatively short time, it moved away from the food and water and moved in the opposite direction to the position of the food and water. From the position after movement, it moved again in the direction of the position where food and water were installed. Such behavior was repeated and did not show a certain behavior pattern.
FIG. 8 shows the activity and trajectory in a 10-minute analysis of an Alzheimer's disease model mouse under open field conditions. It closely resembles the Trajectory pattern of normal littermate mice, and high activities were observed in two locations near the bait and water and near the short side of the cage in the opposite direction to the bait and water (Fig. 8A). In addition, similar to normal litter mice, Trajectory was accumulated in a large amount of Trajectory in the vicinity of food and water and in the vicinity of the cage short side in the opposite direction (FIG. 8B).

1-3. Statistical comparison results between two groups of normal littermate mice and Alzheimer's disease model mice under open field conditions.
As described above, even in the open field condition, five zones were virtually set as in the case where a toy mouse (behavior restriction tool) was installed (With Toy condition) and examined.
Activity analysis results of normal littermate mice group (n = 4) in open field conditions are Zone1 = 25.58 ± 3.06 (mean ± standard deviation), Zone2 = 27.94 ± 3.66, Zone3 = 18.14 ± 3.76, Zone4 = 18.39 ± 1.73 and Zone5 = 9.96 ± 0.54. On the other hand, the activity analysis results of the Alzheimer's disease model mouse group (n = 3) are Zone1 = 22.32 ± 2.28, Zone2 = 29.94 ± 10.83, Zone3 = 24.55 ± 3.53, Zone4 = 17.80 ± 1044, Zone5 = 5.37 ± 1.75 there were.
In the normal littermate mouse group and the Alzheimer's disease model mouse group, a significant difference between the two groups of Zone1, Zone2, Zone3, Zone4, and Zone5 was tested, and no significant difference was found in any of them.
In addition, no difference was found between Trajectory accumulation results between the normal littermate mouse group and the Alzheimer's disease model mouse group (FIGS. 6B and 8B).

2. Results when mouse toy (behavior restriction tool) was additionally installed in the Open field condition (With Toy condition) The food and water (attractant) were installed at the same position as the Open field condition. That is, it was placed at the upper left corner as viewed from above. A mouse toy (behavior restriction tool) was placed between 2.5 cm and 3.5 cm from the right end of the breeding cage.

2-1. Results of behavioral analysis of normal littermate mice under With Toy conditions.
For normal littermate mice, the experiment was started from an intermediate position between the position where food and water, which are instinct stimuli, and the position where the toy for the mouse was installed. Normal littermate mice recognized food and water by looking at the direction of food and water (FIG. 9A). However, a normal littermate mouse recognized food and water, but moved in the opposite direction to its installation position (FIG. 9B), and the mouse toy and the normal breeding cage on the opposite side of the food and water installation position I moved to the area between the walls (Zone4 (Comfortable zone)) and placed myself there (Figs. 9C and D).
Although the experiment in this example was performed in a bright field, it can be assumed that Zone 4 is the most homologous place (reliable zone) of a wild normal mouse that protects itself from the dangers of the outside world in the bright field. Therefore, it is considered that normal littermate mice were placed in Zone4.
FIG. 10 shows the activity and trajectory in a 10-minute analysis of normal littermate mice.
From the analysis result of activity of normal littermate mice for 10 minutes (Fig. 10A), it was found that the activity was highest in Zone4 (safe zone) and low in Zone2 (attractant zone) where food and water boxes were installed. .
Also in the 10-minute integrated image of Trajectory, similarly to the result of Activity, the concentration of Trajectory in Zone 4 (safe zone) was the highest (FIG. 10B). In addition, normal litter mice move along the wall of the normal cage from the photographed image, mounted on the wall of the normal cage of Zone4 (safe zone) (the mouse lifts the hind limb with the forelimb as the wall) It was confirmed that they were doing so-called exploratory behavior to get up. The behavior of such normal littermate mice corresponds to the accumulated images of Trajectory.

2-2. Behavioral analysis results of Alzheimer's disease model mice under With Toy conditions.
The behavioral analysis of the Alzheimer's disease model mouse was also started from an intermediate position between the installation position of the food and water and the installation position of the toy for the mouse, like the normal littermate mouse (FIG. 11A). The Alzheimer's disease model mouse immediately moved in the direction of food and water (FIG. 11B) and placed in Zone 2 (attractant zone) where the food and water box were placed. Moreover, when observed from a behavioral viewpoint, the behavior of sniffing food and feeding food was performed first (FIGS. 10C and D). Eating behavior in the bright field corresponds to the most dangerous time when wild animals are likely to be attacked because attention is paid to food and water in nature. Nevertheless, it has been found that this long-term abnormal feeding behavior is observed in Alzheimer's disease model mice from the beginning of imaging.
FIG. 12 shows the results of 10-minute activity and trajectory of Alzheimer's disease model mice. Activity was high in Zone 2 (attractant zone) where food and water were installed, and was low in Zone 4 (safe zone), which is assumed to correspond to a burrow in nature (FIG. 12A).
In addition, Trajectory showed a high degree of accumulation in Zone 2 (attractant zone) and an image along the toy for mouse in Zone 4 (safe zone) (FIG. 12B). There was no accumulation of trajectory along the wall of the normal rearing cage as seen in normal littermate mice.

2-3. Statistical comparison of activity between normal littermate mice and Alzheimer's disease model mice under With Toy conditions.
The analysis results of With Toy condition in normal litter mice group (n = 4) are Zone1 = 25.63 ± 9.56, Zone2 (attractant zone) = 8.95 ± 8.60, Zone3 = 3.09 ± 1.13, Zone4 (safe zone) = 56.53 ± 17.35, Zone5 = 5.79 ± 3.48.
The analysis results of With Toy in Alzheimer's disease model mouse group (n = 3) are Zone1 = 24.35 ± 1.46, Zone2 (attractant zone) = 48.37 ± 5.24, Zone3 = 3.55 ± 2.03, Zone4 (safe zone) = 17.09 ± 5.45 It was Zone5 = 6.65 ± 4.20.
In Zone2 (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 (safe zone), 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 mice group and Alzheimer's disease model mouse group under With Toy condition.
The comparison of Trajectory was performed based on the comparison of the degree of accumulation of Trajectory strokes for 10 minutes in one mouse.
In the normal littermate mouse group, a large amount of Trajectory in Zone 4 (reliable zone) was normally accumulated on the wall surface of the cage (FIG. 10B). This result shows that normal littermate mice have the same behavioral pattern as wild mice that exist in nature move along the wall. Moreover, the point that the normal littermate mouse performed the mounting which is one of the search behaviors on the wall surface was also consistent with the typical behavior pattern of the wild mouse.
In contrast, Zone 4 (safe zone) Trajectory in the Alzheimer's disease model mouse group does not show accumulation of drawn lines in contact with the wall of the cage, but is accumulated around mouse toys (FIG. 12B). It was different from Trajectory drawn by. That is, in the Alzheimer's disease model mice, a decrease in behavior (such as movement along the wall and mounting search behavior) accompanied by vigilance exhibited by normal littermate mice was observed. This result shows that the trajectory accumulation degree of normal littermate mice and Alzheimer's disease model mice can be distinguished from each other only by comparing the results of Zone4 (safe zone).
In addition, regarding the analysis results at the position where food and water were installed: Zone 2 (attractant zone), a difference was observed between the normal litter mouse group and the Alzheimer's disease model mouse group. That is, the Trajectory of the Alzheimer's disease model mouse group showed a stronger accumulation in Zone 2 (attractant zone) than the normal littermate mouse group. Regarding the accumulation of Trajectory in the other zones, there was no clear difference between the normal littermate mouse group and the Alzheimer's disease model mouse group.

2-5. Correspondence between Activity% and Trajectory results and histopathological analysis results in Alzheimer's disease model mice.
Activity% of With Toy shown in FIG. 13 and pathological histological images of senile plaques and neurofibrils on the hippocampal plane of Alzheimer's disease model mice showing the results of Trajectory accumulation as shown in FIG. 12B were plotted (FIG. 14). . As a result, as shown in the black graph of FIG. 13, in the Alzheimer's disease model mouse in which Activity% in Zone 2 is high and Trajectory accumulation in Zone 2 is recognized, the hippocampus that controls memory and its surroundings There were a lot of neurofibrils around the amygdala, which is the center of emotion, and the elderly group.
From the above, it became clear from the histopathological analysis that Alzheimer's disease model mice exhibiting Alzheimer's disease symptoms show a high activity% in Zone 2 and a strong accumulation of Trajectory.

2-6. Effect on activity and trajectory by administration of compound K that can suppress senile plaque formation and neurofibrillary tangles Alzheimer's disease model mice are treated with candidate compound K or placebo to suppress senile plaque formation and neurofibrillary formation. In addition, placebo was administered to normal littermate mice for 365 days or more, and then, the evaluation method according to the embodiment of the present invention was used to evaluate the presence or absence of abnormal self-issue of these mice (FIG. 15). Regarding the 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, Alzheimer's disease model mice to which Compound K was administered had increased activity in the zone between the attractant zone and the relief zone (FIG. 15, upper center), and behavioral abnormalities characteristic of dementia were normal. Signs of change in behavior were observed. As for Trajectory, the accumulation of Trajectory in Alzheimer's disease model mice administered with Compound K (the lower middle panel in FIG. 15) is intermediate between the accumulation of Alzheimer's disease model mice administered with placebo and the accumulation of normal littermate mice. Characteristic.

In addition, the histopathological analysis of the brains of Alzheimer's disease model mice and normal littermate mice administered with placebo and Alzheimer's disease model mice administered with Compound K was performed. As a result, in the brains of Alzheimer's disease model mice administered with Compound K, the formation sites of senile plaques (upper center diagram in FIG. 16) and neurofibrillary tangles (lower center diagram in FIG. 16) decreased, and normal littermate mice treated with placebo Thus, the therapeutic effect of Compound K could be confirmed (Fig. 16 upper right diagram and lower right diagram).
From the above results, it has been clarified that the method for evaluating experimental animals according to the embodiment of the present invention can be used for screening therapeutic agents for diseases accompanied by self-issued abnormalities.

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

Reference numeral 10; Compartment code 11; Attracting object installation position code 12: Action restriction tool installation position code 20: Detection means code 21; Video means code 22; Control means code 23;

Claims (12)

  A facility for evaluating the behavior of a laboratory animal, comprising a compartment for accommodating a laboratory animal, wherein an attractant and a behavior restriction device are installed in the compartment.   The distance between the compartment partition and the outer surface of the action restriction device, the shortest distance being about 1.0 times to about 1.5 times the lateral length of the body of the experimental animal. The equipment according to claim 1, wherein a tool is installed.   The distance from the outer surface of the behavior restricting device to the outer surface of the attractant, the shortest distance being about 1.5 times to about 2.0 times the lateral length of the body of the experimental animal, The facility according to claim 1 or 2, wherein an attractant is installed.   Detection means for detecting an experimental animal in the compartment, comprising video means for photographing the inside of the compartment, and control means for operating the video means to acquire an image photographed by the video means as a video signal. The equipment according to any one of claims 1 to 3, further comprising a detecting means.   A method for evaluating the presence or absence of an abnormality in the self-issued behavior of a laboratory animal using the facility according to any one of claims 1 to 4, obtained by analyzing a video of the laboratory 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 a laboratory animal using the method for evaluating the presence or absence of an abnormality in self-issued behavior of the laboratory animal according to claim 5. The evaluation method according to claim 6, comprising the following steps (a) to (c).
(A) measuring activity in an attractant zone and / or a relief zone of a control laboratory animal;
(B) a step of measuring the activity in the attract zone and / or the relief zone of the experimental animal to be evaluated, and (c) the attract in which the activity in the attract zone obtained in step (b) is obtained in step (a). If the activity is significantly higher than the activity in the zone and / or 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 that there is a disability The evaluation method according to claim 6, comprising the following steps (a) to (c).
(A) determining the accumulation of Trajectory in the attractant zone and / or the relief zone of the control laboratory animal,
(B) determining the accumulation degree of the Trajectory in the attractant zone and / or the relief zone of the experimental animal to be evaluated; and (c) determining the accumulation degree of the Trajectory in the attractant zone obtained in the step (b) ) Is higher than the degree of trajectory accumulation in the attractant zone obtained in step (b) and / or the degree of trajectory accumulation in the relief zone obtained in step (b) is greater than the degree of trajectory accumulation in the relief zone obtained in step (a). When the experimental animal has a low cognitive function   The disease exhibiting cognitive dysfunction is any of Alzheimer-type dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disorder and intellectual disability based on disease sequelae The evaluation method according to any one of claims 6 to 8, wherein:   10. The method according to claim 5, wherein the experimental animal is a rodent or a small primate.   A method for screening a compound effective for the treatment of a disease accompanied by a self-issued dysfunction using the method according to claim 5.   The disease exhibiting self-issued dysfunction is any of Alzheimer's dementia, cerebrovascular dementia, Lewy body dementia, manic depression, schizophrenia, developmental disorder and intellectual disability based on disease sequelae The method according to claim 11.