WO2014081017A1

WO2014081017A1 - Fear or anxiety measuring system

Info

Publication number: WO2014081017A1
Application number: PCT/JP2013/081548
Authority: WO
Inventors: 高小早川; 令子小早川
Original assignee: 公益財団法人大阪バイオサイエンス研究所
Priority date: 2012-11-22
Filing date: 2013-11-22
Publication date: 2014-05-30
Also published as: JP6218148B2; JPWO2014081017A1

Abstract

　Provided is a method for screening drugs that control emotions (for example preventative or treatment drugs for psychiatric disorders), or aromas that have relaxing effects. A method for screening drugs that control emotions (for example preventative or treatment drugs for psychiatric disorders), or aromas, the method being characterised by comprising: (a) a step for administering a test substance to a test animal or exposing the test animal to the test substance; (b) a step for exposing the test animal to a substance that induces innate fear such as 2-methyl-2-thiazoline or 2, 5-dimethyl-2-thiazoline; (c) a step for measuring at least one value selected from the freezing time, body surface temperature, internal body temperature, and heart rate of the test animal; and (d) a step for selecting a test substance that alters the freezing time, that adjusts a reduction in body surface temperature, that adjusts a reduction in internal body temperature, or that that adjusts a reduction in the heart rate.

Description

Fear or anxiety measurement system

The present invention relates to a method for screening a drug for controlling emotion (for example, a preventive or therapeutic agent for mental illness), a fragrance for inducing a relaxation effect, etc., using a substance that induces congenital fear emotion.

Fear emotion is an instinct acquired by humans and animals to protect themselves from the dangers of foreign enemies and is indispensable for survival. On the other hand, abnormalities in fear affect psychiatric disorders classified as anxiety disorders such as phobia and PTSD (post-traumatic stress disorder). Drugs and fragrances that relieve fear emotions may be used as remedies for mental illnesses or fragrances that induce a relaxing effect. In order to develop such drugs and fragrances, it is effective to analyze the effects of inducing fear emotions on experimental animals or subjects administered the drugs. Therefore, a technique for inducing or measuring fear emotion is useful as a method for screening medicines or fragrances.

There are roughly three types of techniques that have been used so far to induce fear emotions in experimental animals. The first method is a method of inducing fear emotions by combining sensory stimuli such as specific sounds, specific spaces, and specific odors with repellent stimuli such as electric shock and drug injection. . In this method, the learning efficiency varies greatly from individual to individual, so it is difficult to quantitatively induce a specific level of fear emotion. The second method is a technique for inducing a congenital fear emotion using an animal itself that gives fear such as natural enemies, or animal hair, urine, feces, or the like. In this method, fear stimulation is a natural product and it is difficult to maintain a certain quality. Also, with this method, fear emotions cannot be induced with an intensity comparable to the fear acquired in an electric shock. The third method is a method for inducing a congenital fear emotion using a component isolated from the secretions of natural enemies using fear-inducing activity as an index. 2,4,5-Trimethyl-3-thiazoline (TMT), which has been isolated from the fox secretion as the component with the highest fear-inducing activity, has been used. In the method using TMT, fear emotion cannot be induced with an intensity comparable to the fear acquired by electric shock.

The present inventors have discovered that odor molecules containing thiazoline, thiazolidine or thiazole compounds can induce fear emotions to mice with various intensities. By using a series of these odor molecules, it has become possible to steadily induce fear emotions that are stronger than those acquired by an electric shock to fear emotions that are weak.

The present invention provides the following methods.
(1) (a) a step of administering a test substance to a test animal;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) changing the freezing time, adjusting the decrease in body surface temperature, adjusting the decrease in deep body temperature, or selecting a test substance that adjusts the decrease in heart rate, Screening method for drugs to control
(2) The method according to (1), wherein the agent that controls emotion is a preventive or therapeutic agent for mental illness.
(3) (a) a step of administering a test substance to a test animal;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) measuring the deep body temperature of the test animal,
(d) The method according to (1), which is a screening method for a preventive or therapeutic agent for mental illness, comprising a step of selecting a substance that suppresses a decrease in deep body temperature.
(4) The method according to (3), wherein the preventive or therapeutic agent for mental illness is a therapeutic agent for intractable depression.
(5) The method according to any one of (1) to (4), wherein the test animal is a rodent.
(6) (a) exposing the test animal to the test substance;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) A step of changing a freezing time, adjusting a decrease in body surface temperature, adjusting a decrease in body depth temperature, or selecting a test substance that regulates a decrease in heart rate. Screening method.
(7) The method according to (6), wherein the test animal is a rodent.
The present invention also provides the following method.
(8) (a) a step of administering a test substance to a test animal;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) including a step of selecting a test substance that shortens the freezing time, suppresses a decrease in body surface temperature, suppresses a decrease in deep body temperature, or suppresses a decrease in heart rate, A screening method for a prophylactic or therapeutic drug for a disease.
(9) The method according to (8), wherein the test animal is a rodent.
(10) (a) exposing the test animal to the test substance;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) A fragrance comprising a step of selecting a test substance that shortens the freezing time, suppresses a decrease in body surface temperature, suppresses a decrease in deep body temperature, or suppresses a decrease in heart rate. Screening method.
(11) The method according to (10), wherein the test animal is a rodent.

According to the present invention, it is possible to quantitatively induce fear emotions with various intensities by selecting the type of odor molecule used for inducing fear emotions (see FIG. 1). It was clarified that the congenital fear emotion does not acclimatize by repeated stimulation unlike the acquired fear emotion (see FIG. 2). The technology for inducing congenital fear emotion according to the present invention is useful for the purpose of screening for therapeutic agents for mental illnesses such as PTSD, which are difficult to eliminate.

FIG. 1 shows the results of measuring the freezing time (immobility time) of various compounds (Example 1). FIG. 2 shows the freezing time (immobility time) and body surface temperature in mice that induced an acquired fear emotion by anisole and in mice that induced an innate fear emotion by 2MT (2-methyl-2-thiazoline). It is a figure which shows transition (Example 2). FIG. 3 is a diagram showing the results of measuring freezing time, body surface temperature, body deep temperature, and heart rate (Example 3). FIG. 4 is a diagram showing the results of conducting a habituation test for odors that induce fear emotions (Example 4). In the figure, the black line shows the transition of the freezing time when 2MT is sniffed. The white line shows the transition of freezing time when anisole was sniffed in a mouse that had been subjected to fear learning with the smell of anisole. Each value represents mean ± standard error. FIG. 5 shows a schematic diagram of an apparatus used in the screening method of the present invention. FIG. 6 is a line graph showing the rate (%) of freezing time per minute for the groups administered with physiological saline (vehicle), ketamine and memantine (Example 6). FIG. 7A shows the results of a test using 2-methyl-2-thiazoline as a fear odor (Example 7). In FIG. 7A, the mean ± standard error of the freezing time every 10 minutes is shown by a bar graph. FIG. 7B shows the results of a test using 2-methyl-2-thiazoline as the fear odor (Example 7). In FIG. 7B, the mean ± standard error of the difference obtained by subtracting the shrinkage time at each time for each compound by the mean of the shrinkage time of 1-10 minutes is shown as a bar graph. FIG. 8A shows the results of a test using 2,4,5-trimethylthiazole as a fear odor (Example 7). In FIG. 8A, the mean ± standard error of the freezing time every 10 minutes is shown by a bar graph. FIG. 8B shows the results of a test using 2,4,5-trimethylthiazole as the fear odor (Example 7). In FIG. 8B, the mean ± standard error of the difference obtained by subtracting the free time at each time by the average of the free time of 1-10 minutes for each compound is shown by a bar graph. FIG. 9A shows the results of a test using anisole, an acquired fear odor, as a fear odor (Example 7). In FIG. 9A, the mean ± standard error of the freezing time every 10 minutes is shown by a bar graph. FIG. 9B shows the results of a test using anisole, an acquired fear odor, as the fear odor (Example 7). FIG. 9B shows a bar graph showing the mean ± standard error of the difference obtained by subtracting the free time at each time for each compound from the average free time of 1-10 minutes. FIG. 10 shows the results of measuring the effect of psychotropic drugs on Freezing behavior induced by fear odor (Example 8). FIG. 11 shows the results of measuring the effect of a psychotropic drug on the decrease in heart rate and the decrease in deep body temperature induced by fear odor (Example 9). FIG. 12 shows the results of measuring the effects of psychotropic drugs on Freezing behavior, heart rate reduction, and body temperature reduction induced by fear odor (Example 10). FIG. 13 shows the results of measuring the effects on freezing behavior, heart rate, and deep body temperature when memantine or MK801 was injected into the amygdala (Example 11). FIG. 14 shows the results of measuring the effect of psychotropic drugs on the decrease in body surface temperature induced by fear odor (Example 12). FIG. 15 shows the results of measuring changes in the deep body temperature of pigs with respect to fear odor (Example 15). FIG. 16 shows the results of measuring changes in pig body surface temperature with respect to fear odor (Example 16). FIG. 17 shows the results of observing the effects of psychotropic drugs on freezing behavior (a) and fluency (b) of pigs induced by fear odor (Example 17).

恐怖 Fear emotions can be induced innately or acquiredly by various sensory inputs. For example, if you visually sense that you are in a dangerously high place, you will feel fear (fear of height). If you sense that you are confined in a narrow space where you cannot move, you will feel fear (closed fear). Herbivores feel fear when they sense the smell of carnivores, natural enemies, by smell (the fear of natural enemy odors). These fears are innate fears that humans and animals have born to avoid dangerous things (Reference 1: NatureNvol.450, p503-508 (2007)). On the other hand, there are also acquired fears, as shown in the example of feeling a fear at a certain place after experiencing a dangerous experience. In studies conducted so far, it is considered that a variety of sensory inputs activate the fear center of the brain to generate a common fear emotion. On the other hand, the present inventors have clarified for the first time that fear emotions show different types of physiological responses depending on the type of sensory input induced and whether the induced method is congenital or acquired (FIG. 3). reference). In particular, the congenital fear emotions for odor molecules that can be triggered for the first time by the present invention are processed by a different neural mechanism than the acquired fear emotions that can be triggered by conventional techniques, resulting in different physiological responses. It is considered a completely new fear in the sense that it accompanies.

後 Acquired fear emotions that could be triggered by conventional techniques can be quantitatively measured using freezing behavior, stress hormone secretion, etc. as indicators. The congenital fear emotions induced using the odor molecules of the present invention cannot be distinguished from acquired fear emotions as long as conventional indices such as freezing behavior and stress hormone secretion are used. However, the present inventors have found that congenital fear emotions due to odor molecules, unlike acquired fear emotions, are accompanied by a decrease in body surface temperature, a decrease in deep body temperature, and a decrease in heart rate. Therefore, by using the decrease in body surface temperature, deep body temperature or heart rate as an index, congenital and acquired fear emotions can be measured separately (see FIG. 3).

According to the present invention, by using a specific odor molecule, it is possible to induce an innate fear emotion that has completely different characteristics from the acquired fear emotion that can be induced by conventional techniques.

As shown in FIG. 1, the use of odor molecules such as 2-methyl-2-thiazoline is 10 times more intense than the fear emotions that could be induced by conventional techniques using TMT. It is possible to induce innate fear emotions.

In addition, it has been difficult to quantitatively induce innate fear emotions of various intensities with the conventional technique, but in the present invention, by using different types of odor molecules shown in FIG. It is possible to induce fear emotions.

In the prior art, it was impossible to distinguish between congenital and acquired fear emotions, but by using at least one selected from body surface temperature, body temperature, and heart rate as an index, it is possible to distinguish between congenital and acquired fear emotions. It is possible to measure the fear emotion.

Hereinafter, the screening method of the present invention will be described in detail.
As test animals, rodents such as mice, rats, guinea pigs, and hamsters, rabbits, pigs, and other mammals can be used. Preferably, a mouse can be used. There are no particular restrictions on the sex, age, and weight of the test animal.

In the method for screening a drug for controlling emotion according to the present invention, the order of (a) administering a test substance to a test animal and (b) exposing the test animal to a substance that induces a congenital fear emotion is limited. Instead, step (b) may be performed after step (a), or step (a) may be performed after step (b).

In the screening method for a preventive or therapeutic agent for mental illness of the present invention, the sequence of (a) administering a test substance to a test animal, and (b) exposing the test animal to a substance that induces congenital fear emotions. Is not limited, and step (b) may be performed after step (a), or step (a) may be performed after step (b).

Similarly, in the fragrance screening method of the present invention, the order of (a) exposing a test animal to a test substance and (b) exposing the test animal to a substance that induces a congenital fear emotion is limited. Instead, step (b) may be performed after step (a), and step (a) may be performed after step (b).

Administration of the test substance can be performed by oral administration, intravenous administration, intraperitoneal administration, transdermal administration, etc. according to the type of the test substance. When the test substance is administered orally, the test substance can be dissolved in water or an organic solvent before administration. The dose of the test substance can be appropriately changed depending on the type of test animal, the type of test substance, and the administration method. For example, when a test substance is orally administered using a mouse, usually about 1 μl to about 5 ml, preferably about 50 μl to 500 μl of the test substance is orally administered.

After the test substance is administered to the test animal, the time until the test animal is exposed to the substance that induces innate fear emotion can be appropriately changed according to the type of test animal and the test substance administration method. For example, when a test substance is orally administered using a mouse, the test animal is added to a substance that induces a congenital fear emotion 1 minute to 2 years, preferably 5 minutes to 1 hour after oral administration of the test substance. To be exposed.

After the test animal is exposed to a substance that induces a congenital fear emotion, the time until the test substance is administered to the test animal can be appropriately changed according to the type of test animal and the test substance administration method. For example, when a test substance is orally administered using a mouse, the test substance is administered to the test animal 1 minute to 2 years after exposure to a substance that induces congenital fear emotion, preferably 5 minutes to 1 hour. Administer.

A substance that induces a congenital fear emotion is usually exposed to a test animal in an amount of about 1 μmol to 30 mmol or 0.1 to 1000 ppm, preferably about 30 μmol to 3 mmol or 1 to 100 ppm.

In the case of screening fragrances, the time for exposing the test animal to the test substance is usually 10 seconds to 2 years, preferably 10 seconds to 1 day. The test substance is usually exposed to the test animal in an amount of about 1 nmol to 30 mmol or 0.1 to 1000 ppm, preferably 1 μmol to 3 mmol or 1 to 100 ppm.

When screening a fragrance, the time from exposure of a test animal to a test substance to exposure of the test animal to a substance that induces an innate fear emotion can be appropriately changed depending on the type of test animal. For example, when using a mouse, the test animal is exposed to a substance that induces congenital fear emotions after 1 minute to 2 years, preferably 1 minute to 1 hour, after exposure to the test substance.

When screening perfumes, the time from exposure of a test animal to a substance that induces a congenital fear emotion to exposure of the test animal to the test substance can be appropriately changed depending on the type of test animal. For example, when using a mouse, the test animal is exposed to the test substance 1 minute to 2 years after exposure to the substance that induces congenital fear emotions, preferably 1 minute to 1 hour.

The freezing time is defined as a time during which the test animal is stationary for a certain time (for example, 2 seconds) or more. The freezing time of the test animal can be measured using, for example, a behavioral analysis camera or an infrared sensor.

The body surface temperature of the test animal can be measured using, for example, a thermography camera for body surface temperature measurement.

Body deep temperature is defined as the temperature inside the body. The deep body temperature of the test animal can be measured, for example, by embedding a deep body temperature measurement transmitter in the test animal.

The heart rate of the test animal can be measured, for example, by embedding a heart rate measurement transmitter in the test animal.

In the screening method of the present invention, at least one index selected from freezing time, body surface temperature, body depth temperature, and heart rate may be measured. Preferably, the freezing time, body surface temperature, deep body temperature, and heart rate are all measured.

Examples of drugs that control emotions that can be screened by the method of the present invention include preventive or therapeutic drugs for mental illness. Mental illnesses include, but are not limited to, anxiety disorders, depression, refractory depression, depression, bipolar disorder, PTSD, schizophrenia and the like. Examples of preventive or therapeutic agents for mental illness include anxiolytics, antidepressants, refractory depression, mood stabilizers, PTSD, and schizophrenia. It is not limited to.

Examples of the fragrances that can be screened by the method of the present invention include fragrances that have effects of inducing a relaxing effect, such as sedation, stress reduction, and sleep promotion.

When used as a screening system for a drug for controlling emotion, a group of test animals to which a candidate substance for a drug for controlling emotion is administered in advance (hereinafter referred to as a test substance-administered group) and a group of test animals to which no test substance is administered (Hereinafter referred to as the control group), and changes in behavior (shrinking time) and physiological responses (body surface temperature, body temperature, heart rate) when fear emotions are induced by odor molecules for each group. To analyze. In order to eliminate the possibility that a candidate drug that controls emotions does not specifically inhibit fear emotions, the behavior of odor molecules that do not induce congenital fear emotions (for example, anisole) Changes in physiological responses are also analyzed.

Analyzing multiple individuals (at least 3 animals, preferably 8 animals or more) for each of the control group and the test substance administration group, and when the change in the freezing time (shortening or increasing, preferably shortening) is found to be statistically significant The substance can be selected as an agent that controls emotion.

When multiple individuals (at least 3, preferably 8 animals) are analyzed for each of the control group and the test substance administration group, and the adjustment (suppression or enhancement, preferably inhibition) of the decrease in body surface temperature is statistically significant In addition, the test substance can be selected as a drug for controlling the emotion.

When multiple individuals (at least 3, preferably 8 animals) are analyzed for each of the control group and the test substance administration group, and the adjustment (suppression or enhancement, preferably inhibition) of the decrease in body temperature is statistically significant In addition, the test substance can be selected as a drug for controlling the emotion.

When the control group and the test substance administration group are each analyzed by a plurality of individuals (at least 3 animals, preferably 8 animals or more), and the adjustment (suppression or enhancement, preferably inhibition) of heart rate reduction is statistically significant The test substance can be selected as a drug for controlling the emotion.

When used as a screening system for preventive or therapeutic drugs for mental illnesses such as anxiolytic drugs, a group of test animals to which candidate substances for preventive or therapeutic drugs such as anxiolytic drugs have been administered in advance (hereinafter referred to as test substance administration groups) And a group of test animals not administered the test substance (hereinafter referred to as a control group), and behavior (shrinking time) and physiological response (when the fear emotion is induced by odor molecules for each group) Analyze changes in body surface temperature, deep body temperature, heart rate). In order to eliminate the possibility that candidate substances for the prevention or treatment of mental disorders such as anxiolytic drugs do not specifically inhibit fear emotions, odor molecules that do not induce congenital fear emotions (eg, anisole) We also analyze changes in behavior and physiological responses when they are generated.

Analyzing multiple individuals (at least 3 animals, preferably 8 animals or more) for each of the control group and the test substance administration group, and when the change in the freezing time (shortening or increasing, preferably shortening) is found to be statistically significant The substance can be selected as a preventive or therapeutic agent for mental illness.

When multiple individuals (at least 3, preferably 8 animals) are analyzed for each of the control group and the test substance administration group, and the adjustment (suppression or enhancement, preferably inhibition) of the decrease in body surface temperature is statistically significant In addition, the test substance can be selected as a preventive or therapeutic agent for mental illness.

When multiple individuals (at least 3, preferably 8 animals) are analyzed for each of the control group and the test substance administration group, and the adjustment (suppression or enhancement, preferably inhibition) of the decrease in body temperature is statistically significant In addition, the test substance can be selected as a preventive or therapeutic agent for mental illness.

In particular, when a plurality of individuals (at least 3, preferably 8 or more) are analyzed in each of the control group and the test substance administration group, and the suppression of a decrease in body temperature is statistically significant, the test substance is antidepressed. It can be selected as a drug (including a treatment for refractory depression).

When the control group and the test substance administration group are each analyzed by a plurality of individuals (at least 3 animals, preferably 8 animals or more), and the adjustment (suppression or enhancement, preferably inhibition) of heart rate reduction is statistically significant The test substance can be selected as a preventive or therapeutic agent for mental illness.

When measuring the relaxation effect of fragrances, etc., the fragrances to be analyzed are acted on in advance by sniffing the test animal or placed in a soundproof box, etc. Changes in fear response (shrinking time, body surface temperature, body temperature, heart rate) when no action is taken are quantitatively analyzed.

A group of test animals not exposed to the test substance (hereinafter referred to as the control group) and a group of test animals exposed to the test substance (hereinafter referred to as the test substance exposure group) (each at least 3, preferably 8 animals or more) Analyzing and selecting a test substance as an effective fragrance if the change in shrinkage time (shortening or increasing, preferably shortening) is found to be statistically significant.

When multiple individuals (at least 3, preferably 8 or more) are analyzed for each of the control group and the test substance exposure group, and the adjustment (suppression or enhancement, preferably suppression) of the decrease in body surface temperature is statistically significant In addition, the test substance can be selected as an effective fragrance.

When multiple individuals (at least 3, preferably 8 or more) are analyzed for each of the control group and the test substance exposure group, and the adjustment (suppression or enhancement, preferably suppression) of the decrease in body temperature is statistically significant In addition, the test substance can be selected as an effective fragrance.

Analyzing multiple individuals (at least 3 animals, preferably 8 animals or more) in each of the control group and the test substance exposure group, and if the control (suppression or enhancement) of heart rate reduction is found to be statistically significant, It can be selected as an effective fragrance.

Examples of substances that induce congenital fear emotion include compounds described as active ingredients of animal repellents in WO2011 / 096575, 2,4,5-trimethylthiazole.

Preferred examples of substances that induce innate fear emotions include formulas (A), (B), (C), (F), (G), and (H):

(Where
R ₁ , R ₂ and R ₃ are each independently hydrogen, halogen atom, C _1-6 alkyl group, C _1-6 haloalkyl group, C _1-6 alkoxy group, C _1-6 haloalkoxy group, formyl group, C _1-6 alkyl-carbonyl group, carboxyl group, C _1-6 alkoxycarbonyl group, thiol group, C _1-6 alkylthio group, amino group, C _1-6 alkylamino group, di (C _1-6 alkyl) amino A group, —NR ₄ COR ₅ or an oxo group,
R ₄ and R ₅ each independently represent hydrogen or a C _1-6 alkyl group.
However, in formula (A), R ₁ and R ₂ are not oxo groups; in formula (B) and formula (G), R ₁ is not an oxo group; in formula (C), R ₁ and R ₃ are combined. To form an oxo group. )
And a compound selected from 2,4,5-trimethylthiazole or a salt thereof.

In the formulas (A), (B), (C), (F), (G) and (H),
R ₁ represents hydrogen, a halogen atom (eg, bromine atom), a C _1-6 alkyl group (eg, methyl, ethyl) or a C _1-6 alkylthio group (eg, methylthio),
R ₂ represents hydrogen or a C _1-6 alkyl group (eg, methyl),
A compound or a salt thereof in which R ₃ represents hydrogen or a C _1-6 alkyl group (eg, methyl) is more preferable.

Preferable examples of the compound of the formula (A) include 2-methylthiazole, 2-ethylthiazole, 2-bromothiazole, 4-methylthiazole, 2,4-dimethylthiazole and the like.

Preferable examples of the compound of the formula (B) include 2-methyl-2-thiazoline, 2-methylthio-2-thiazoline, 4-methyl-2-thiazoline, 2,4-dimethyl-2-thiazoline and the like.

Preferred examples of the compound of the formula (C) include thiazolidine, 2-methylthiazolidine, 2,2-dimethylthiazolidine, 4-methylthiazolidine, 2,4-dimethylthiazolidine and the like.

Preferred examples of the compound of formula (F) include thiomorpholine.

Preferable examples of the compound of formula (G) include 2,5-dimethyl-2-thiazoline or 5-methyl-2-thiazoline.

Preferred examples of the compound of formula (H) include 5-methylthiazolidine.

2-methyl-2-thiazoline is particularly preferred as a substance that induces innate fear emotions.

The above-mentioned compounds can be used commercially, or can be obtained by a method known per se.

The salt of the compound according to the present invention includes any pharmaceutically acceptable salt, for example, alkali metal salts such as sodium salt and potassium salt; magnesium salt and calcium salt, etc. Alkaline earth metal salts; ammonium salts such as dimethylammonium salt and triethylammonium salt; inorganic acid salts such as hydrochloride, perchlorate, sulfate and nitrate; organic acids such as acetate and methanesulfonate Examples include salt.

The present invention also provides a screening apparatus used in the screening method of the present invention.

The screening apparatus of the present invention comprises:
A rearing cage containing the test animal,
An odor molecule generator connected to the breeding cage,
Detection means for detecting the behavior of the test animal housed in the breeding cage, and means for measuring at least one selected from the body surface temperature, the body depth temperature and the heart rate of the test animal housed in the breeding cage It is characterized by providing.

The apparatus of the present invention can be used for screening for preventive or therapeutic agents for mental illnesses or fragrances.

Odor molecule generator can generate odor molecules in a breeding cage through a nozzle.

As a detection means for detecting the behavior of the test animal, a behavior analysis camera, an infrared sensor, or the like can be used. Preferably, a behavior analysis camera can be used.

As a means for measuring the body surface temperature of the test animal, a thermographic camera for body surface temperature measurement can be preferably used.

As a means for measuring at least one selected from the body temperature and heart rate of the test animal, the heart rate / body temperature measurement is installed adjacent to the breeding cage by embedding a heart rate / body temperature measurement transmitter in the test animal. Examples of the receiver include means for receiving measurement data of heart rate and deep body temperature transmitted from the transmitter.

The odor molecule generator, the detection means for detecting the behavior of the subject animal, and the means for measuring at least one selected from the body surface temperature, the body depth temperature and the heart rate of the subject animal are connected to the control / analysis computer. Can be controlled.

The breeding cage is preferably housed in a soundproof box. The detection means for detecting the behavior of the subject animal and the means for measuring at least one selected from the body surface temperature, the body depth temperature and the heart rate of the subject animal are preferably connected to a soundproof box. An exhaust fan, lighting, etc. can be installed in the soundproof box.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the Examples are not intended to limit the present invention.

Example 1
A breeding cage was placed in the draft and a mouse was placed. Subsequently, the filter paper on which 270.6 μmol of odor molecules were dropped was placed in a breeding cage. During the subsequent 20 minutes, the percentage of time during which the mouse showed freezing behavior was calculated using video analysis software. Freezing time was defined as the time during which the mouse was stationary for 2 seconds. The experiment was repeated using 8 or more mice for one kind of odor molecule, and the average and standard error were calculated. The results are shown in FIG.

Example 2
In order to measure the difference in physiological responses associated with acquired and congenital fear emotions, the body surface temperature of mice that induced congenital and acquired freezing behavior was measured using a thermographic camera. In order to induce acquired freezing behavior, the mice were sniffed randomly with the scent of control Eugenol and the anisole scent, which is related learning, and only when the scent of anisole was sniffed. I received an electric shock. By this operation, the mouse learns the anisole smell and the electric shock in association with each other, and when the smell of the anisole is smelled, an acquired freezing action can be induced.

A breeding cage was placed in the draft and a mouse was inserted. At 0 minutes after the start of the experiment, filter paper on which no odor molecule was dropped was placed in a cage. After removing odorless filter paper from the cage at 20 minutes, filter paper impregnated with eugenol, a control odor molecule, was placed in the cage. Remove the filter paper soaked with eugenol at 40 minutes and add 270.6 mol of 2MT (2-methyl-2-thiazoline), an anisole that induces an acquired fear emotion, or 2MT (2-methyl-2-thiazoline) that induces an acquired fear emotion In a cage.

Fig. 2 shows changes in freezing time (immobility time) and body surface temperature in a mouse that induced an acquired fear emotion by anisole and a mouse that induced an innate fear emotion by 2MT.

Both the mice that induced an acquired fear emotion by anisole and the mice that induced the congenital fear emotion by 2MT show the same time course and frequency of shrinkage, but the mouse that induced the congenital fear emotion by 2MT Only a decrease in body surface temperature was observed. Therefore, the decrease in body surface temperature can be used as an index for specifically measuring innate fear emotions.

Example 3
The odor of 2-methyl-2-thiazoline (2MT), 2,5-dimethyl-2-thiazoline (2,5DMT), or 2,4,5-trimethyl-3-thiazoline (TMT) is shown in Example 1. By smelling with the method, innate fear emotions were induced. Acquired fear emotion was induced by smelling anisole after learning the anisole odor and electric shock in a related manner by the method shown in Example 2. By placing the mouse into a plastic tube with a diameter of 2.9 cm, it was made incapable of moving, thereby inducing a congenital fear emotion by restraint. The graph of FIG. 3 shows the free time of 20 minutes after the presentation of the fear stimulus, the body surface temperature, the body depth temperature, and the mean ± standard error of the heart rate.

A. The level of freezing action using 2MT or 2,5DMT is comparable to the freezing-induced freezing action. Compared to these levels of freezing behavior, the level of freezing behavior induced using the known TMT is only one tenth. Therefore, by using the odor molecule of the present invention, it is possible to induce a high-frequency freezing action that requires an operation for performing learning related to an electronic shock by an operation that only smells the odor molecule.

The body surface temperature of the mouse under the four conditions shown in b. A and the conditions in which congenital fear emotions were induced by restraint was analyzed using a thermographic camera. The body surface temperature decreased by about 2 ° C for fear emotions congenitally induced using 2MT or 2,5DMT, and by about 0.6 ° C for fear emotions congenitally induced using TMT. In the acquired emotional fear, the decrease in body surface temperature is only about 0.2 ° C. In the congenital fear emotion due to restraint, the body surface temperature decreases by about 5 ° C. From the above experimental results, it became clear for the first time that body surface temperature can be used as an index for measuring congenital fear emotions.

C, d. Deep body temperature and heart rate were measured using a wireless body temperature and heart rate measuring device embedded in the body.

C. 2MT or 2,5DMT congenital fear emotions induced by about 2 ° C, and TMT congenitally induced fear emotions decreased by about 0.2 ° C. On the other hand, the deep body temperature rose by about 0.2 ° C. in the fear emotion that was acquired afterward. In the congenital fear emotion due to restraint, the deep body temperature decreased by about 1.4 ° C. From the above results, it became clear for the first time that a decrease in body temperature can be used as an index for measuring congenital fear emotions.

D. 心拍 2MT or 2,5DMT congenital fear emotions reduced heart rate by about 250 bpm (beat 、 permin), congenitally induced fear emotions using TMT by about 70 bpm. The heart rate decreased by about 40 bpm in congenitally induced fear emotions by restraint. In the acquired emotional fear, the heart rate decreased only by about 10 bpm. From the above results, it became clear for the first time that a decrease in heart rate can be used as an index for measuring congenital fear.

Example 4
2-methyl-2-thiazoline (black), which induces congenital fear, and anisole (white), which has acquired acquired fear by electric shock and related learning, continue to smell once a day for 8 consecutive days The freezing time (immobility time) was compared. After the mouse was placed in a breeding cage placed in a draft, a filter paper cut into a 1.5 cm square and impregnated with odor molecules (270.6 μmol) was placed in the cage. The freezing time of the mouse (N = 8) in the measurement time of 20 minutes was measured. The results are shown in FIG. The Student-t test was performed on the freezing time on the first day and the freezing time on and after the second day. * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001, indicating that there is a significant difference. It was found that the frequency of freezing caused by the smell of acquired fear gradually decreased (erasing fear), but the frequency of congenital freezing caused by “fear odor” gradually increased. (Enhancing fear). From this experimental result, it became clear that innate and acquired fear emotions have distinctly different properties in terms of ease of erasure.

Example 5
FIG. 5 shows a schematic diagram of an example of the screening apparatus of the present invention. Place the rearing cage (d) in the soundproof box (a). The soundproof box is equipped with a camera for freezing behavior analysis (b), a thermography camera for body surface temperature measurement (c), an exhaust fan (g), illumination (h), and a heartbeat / deep body temperature measurement receiver (i). To do. Various odor molecules shown in FIG. 1 are generated from the bottom of the breeding cage through a nozzle (f) connected to the odor molecule generator (e). The odor molecule generator and various analyzers are controlled by a control / analysis computer (k). Before starting the measurement of fear emotion, a test animal in which a heart rate / body temperature measuring transmitter (j) is embedded is placed in a breeding cage (d). As test animals, rodents such as mice, rats, guinea pigs, hamsters, rabbits, and other mammals can be used. Odor molecules are generated at a specific time, and the fear emotion is quantitatively measured by measuring at least one selected from the freezing time, body surface temperature, body temperature, and heart rate.

Example 6
Saline (vehicle), ketamine (5 mg / kg), and memantine (25 mg / kg) were each intraperitoneally administered to mice (N = 6), and 30 minutes later, the mice were placed in a test cage. Moved. In the test cage, 0-10 minutes had no odor, 11-20 minutes had Eugenol (270.6 μmol) odor as a control, and 21-40 minutes had terrible odor by the method shown in Example 1. . In this example, 2-Methyl-2-thiazoline (270.6 μmol) was used as the fear odor. The freezing time for 40 minutes after moving the mouse to the test cage was measured, and the percentage (%) of the freezing time per minute was shown as a line graph (FIG. 6). In the group administered with ketamine, there was no change compared with the vehicle, but in the group administered with memantine, freezing action against 2-methyl-2-thiazoline was suppressed.

Example 7
Saline (vehicle), diazepam (Diazepam), tandospirone (Tandospirone), fluoxetine (Dluxetine), clozapine (Clozapine), risperidone (Risperidone), MK801, ketamine (CPetamine), CP101,606, riluzole Riluzole) and memantine were administered to mice (N = 6-8). MK801 and memantine were administered by intraperitoneal injection, and other compounds were administered orally. Mice were transferred to the test cage after about 1 hour for oral administration and about 30 minutes for intraperitoneal injection. In the test cage, as in Example 6, 0-10 minutes had no odor, 11-20 minutes had eugenol (270.6 μmol) as a control, and 21-40 minutes had a fear odor, 2-methyl-2-thiazoline. (270.6 μmol) was presented and the freezing behavior of the mice was observed. In FIG. 7A, the mean ± standard error of the freezing time every 10 minutes is shown in FIG. 7B. In FIG. 7B, the average ± standard error of the difference obtained by subtracting the freezing time at each time by the average of the freezing time of 1-10 minutes is shown for each compound. It showed in. The Student-t test was performed on the freezing time when the vehicle and each compound were given at each time. * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001, indicating that there is a significant difference.

Saline (vehicle), diazepam, tandospirone, fluoxetine, duloxetine, clozapine, risperidone, MK801, ketamine, and memantine Compounds were administered to mice (N = 6-10) orally or by intraperitoneal injection. MK801 and memantine were administered by intraperitoneal injection, and other compounds were administered orally. Mice were transferred to the test cage after about 1 hour for oral administration and about 30 minutes for intraperitoneal injection. In the test cage, as in Example 6, 0-10 minutes had no odor, 11-20 minutes had eugenol (270.6 μmol) as a control, 21-40 minutes had a fear odor, and the mouse's freezing behavior was observed. did. In this example, 2,4,5-trimethylthiazole (270.6 μmol) was used as a fear odor. In FIG. 8A, the mean ± standard error of the freezing time every 10 minutes is shown in FIG. 8B. In FIG. 8B, the average ± standard error of the difference obtained by subtracting the freezing time at each time by the average of the freezing time of 1-10 minutes is shown for each compound. It showed in. The Student-t test was performed on the freezing time when the vehicle and each compound were given at each time. * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001, indicating that there is a significant difference.

Saline (vehicle), diazepam, tandospirone, duloxetine, clozapine, risperidone, and MK801 are given orally or intraperitoneally to mice (N = 6-10). Administered by injection. MK801 was administered by intraperitoneal injection, and other compounds were orally administered. Mice were transferred to the test cage after about 1 hour for oral administration and about 30 minutes for intraperitoneal injection. In the test cage, as in Example 6, 0-10 minutes had no odor, 11-20 minutes had eugenol (270.6 μmol) as control, and 21-40 minutes had fear odor. Was observed. In this example, the mouse learned in advance that when it gave the eugenol odor, it did not give an electric shock, but only when it gave the odor of anisole (270.6 μmol). The anisole which is the smell of the acquired fear was used as a fear odor. In FIG. 9A, the mean ± standard error of the freezing time every 10 minutes is shown in FIG. 9B. In FIG. 9B, the mean ± standard error of the difference obtained by subtracting the freezing time at each time by the average of the freezing time of 1-10 minutes for each compound is a bar graph. It showed in. The Student-t test was performed on the freezing time when the vehicle and each compound were given at each time. * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001, indicating that there is a significant difference.

Example 8
a By learning the relationship between electrical foot shock (FC) and spice-derived odor molecules (Anisole), the acquired Freezing behavior can be induced by smelling anisole (Anis-FC).

b Smell

2,4,5-trimethyl-thiazole (245TMT) can induce a weak level of innate Freezing behavior (Innate-low). c Smelling 2-methyl-2-thiazoline (2MT) can induce a strong level of innate freezing behavior (Innate-high).

¡Freezing behavior associated with three types of fear emotions was induced in mice by the methods a, b, and c. The effects of various psychotropic drugs on these three types of Freezing behavior were measured quantitatively. The results are shown in FIG. The psychotropic drugs were administered by oral administration (Oral) or intraperitoneal injection (IP) (N = 6-8). The psychotropic drugs used were the anti-anxiety drugs Diazepam (2 concentrations), antidepressants (Fluoxetine), duloxetine, and atypical antipsychotics (risperidone) Clozapine), antagonists of NMDA receptors (Memantine, MK801, Ketamine, Traxoprodil). Diazepam, fluoxetine, duloxetine, risperidone, and clozapine were administered orally, and memantine, ketamine, and traxoprodil were administered intraperitoneally. The dose of each psychotropic drug is also shown in FIG.

The level of fear emotion can be quantitatively measured by the accumulated time of Freezing action. As compared with the condition in which physiological saline was administered as a control, the accumulated time of freezing behavior that increased or decreased was shown in the graph of FIG. 10 (ΔFreezing%). An increase in Freezing indicates an increased level of fear emotion. When the psychotropic drug administration significantly increased the Freezing integration time compared to the control condition, it was indicated by the # symbol (Student's t-test #: P <0.05, ##: P <0.01, ## #: P <0.001). Conversely, when the psychotropic drug administration significantly reduced the Freezing integration time compared to the control condition, it was indicated by * (Student's t-test- *: P <0.05, **: P <0.01. , ***: P <0.001). With regard to acquired FreezingL (Learned), diazepam (0.1 / mg / kg), fluoxetine, risperidone (0.04 / mg / kg), and memantine administration significantly reduced the Freezing cumulative duration. In addition, administration of diazepam (3 mg / kg) significantly increased the freezing accumulation duration. Regarding low levels of congenital Freezing® (Innate-Low), administration of diazepam® (3 mg / kg), risperidone® (0.3 mg / kg), clozapine, memantine, and MK801 significantly increased Freezing integration time. For high levels of innate Freezing® (Innate-High), administration of diazepam® (3 mg / kg) and risperidone® (0.3 μgm / kg) significantly increased the freezing integration time. Conversely, administration of memantine and MK801 significantly reduced the freezing cumulative duration.

From the above results, it can be concluded that the effects of psychotropic drugs on the freezing behavior of the three types of acquired Freezing, low-level innate Freezing, and high-level innate Freezing are different. Therefore, it is possible to classify and evaluate the efficacy of psychotropic drugs by inducing different types of Freezing using the fear odor.

Example 9
Under conditions that induce a high level of innate fear response by utilizing odor molecules that induce innate fear, in addition to inducing freezing behavior, heart rate (ΔHeart rate) and deep body temperature ( ΔCore temperature) is also triggered at the same time. By using physiological responses specific to these high-level innate fears as an index, the efficacy of psychotropic drugs can be evaluated. We analyzed the effects of various psychotropic drugs on the decrease in heart rate induced by 2-methyl-2-thiazoline (2MT) in mice and the decrease in body temperature. The results are shown in FIG. The type, dosage and administration method of the psychotropic drug are the same as in Example 8 (N = 6-8). Saline was administered to the control group. If psychotropic drug administration significantly enhanced the decrease in heart rate or deep body temperature compared to control conditions, it was indicated by the # symbol (Student's t-test #: P <0.05, ##: P <0.01, ###: P <0.001). Conversely, if the psychotropic drug significantly reduced the decrease in heart rate or deep body temperature compared to the control condition, it was indicated by the * symbol (Student's t-test *: P <0.05, **: P <0.01, ***: P <0.001). Administration of diazepam (3 mg / kg), memantine, MK801, ketamine, and traxoprodil alleviated 2MT-induced heart rate reduction. Using declining heart rate as an indicator, these psychotropic drugs suggested that they alleviated fear emotions. Administration of diazepam (3 mg / kg), MK801, ketamine and traxoprodil alleviated the 2MT-induced decrease in body temperature. Conversely, administration of risperidone (0.3 mg / kg) and memantine enhanced the 2MT-induced decrease in body temperature. Freezing behavior induced by 2MT, decreased heart rate, and decreased body temperature have different response specificities for psychotropic drugs. Therefore, by analyzing the effects of behaviors and physiological responses induced by 2MT on psychotropic drugs, the effects of psychotropic drugs can be classified and evaluated with unprecedented accuracy.

Example 10
NMDA receptor antagonists against 2-zing-2-thiazoline-induced freezing behavior, reduced heart rate (ΔHeart rate), decreased core temperature (ΔCore temperature) ( The effects of memantine, MK801, ketamine, and traxoprodil) were analyzed.

The experimental procedure is shown below. Mice were administered psychotropic drugs or control saline by intraperitoneal injection (N = 6-8). The dose of the psychotropic drug is shown in FIG. Subsequently, the mice were transferred to a test cage and acclimated. Unscented filter paper was placed in the test cage (0-10 min). Subsequently, a filter paper soaked with a spice odor (Eugenol), which is a control odor that does not induce innate fear emotions, was placed in a test cage (10-20 min). After removing the filter paper soaked with eugenol, the filter paper soaked with 2-methyl-2-thiazoline (2MT) was placed in the test cage (20-40 min). We analyzed the effects of psychotropic drugs on Freezing behavior every 5 minutes, decreased heart rate, and decreased body temperature. The results are shown in FIG. The number of fear emotions was significantly increased (increased freezing behavior, increased heart rate and decreased body temperature) compared to the conditions administered with saline (Student's t-test # : P <0.05, ##: P <0.01, ###: P <0.001). Conversely, compared to the conditions in which physiological saline was administered, the level of fear emotion was significantly increased (decreased freezing behavior, alleviation of heart rate and deep body temperature decrease) by the symbol * (Student's t -test *: P <0.05, **: P <0.01, ***: P <0.001). The pharmacological effects of memantine, MK801, ketamine and traxoprodil could be measured separately.
Ketamine and traxoprodil have been reported to have therapeutic effects on intractable depression. In contrast, memantine has been reported to have no therapeutic effect on depression, and MK801 has been reported to have various side effects. It was found that memantine enhances the 2MT-induced decrease in deep body temperature, and MK801 has the side effect of increasing the deep body temperature in the absence of odor or in the condition of smelling control. On the other hand, ketamine and traxoprodil do not affect the deep body temperature when smelling no smell or control, and specifically suppress the decrease in deep body temperature when sniffing 2MT. Became clear. Therefore, the effect of a drug on refractory depression, other mental illnesses, etc. can be specifically measured by using 2MT-induced temperature drop in the deep body as an index.

Example 11
The effects of injecting memantine and MK801 into the amygdala were analyzed. The amygdala is thought to be the center of emotions such as fear. A guide cannula was inserted into the mouse amygdala and fixed. After surgery, the mice were rested for about one week. Thereafter, memantine or MK801 was injected through the guide cannula. Saline was injected as a control. Using the conditions in which physiological saline was injected into the amygdala as a control, we analyzed the effects on freezing behavior, heart rate, and deep body temperature when memantine or MK801 was injected into the amygdala.

The experimental procedure is shown below. Mice were administered psychotropic drugs or control saline (N = 6-8). The dose of the psychotropic drug is shown in FIG. Subsequently, the mice were transferred to a test cage and acclimated. Unscented filter paper was placed in the test cage (0-10 min). Subsequently, a filter paper soaked with spice odor (eugenol), a control odor that does not induce congenital fear emotions, was placed in a test cage (10-20 min). After removing the filter paper soaked with eugenol, the filter paper soaked with 2-methyl-2-thiazoline soot (2MT) was placed in a test cage (20-40 min). We analyzed the effects of psychotropic drugs on Freezing behavior every 5 minutes, decreased heart rate, and decreased body temperature. The results are shown in FIG. Compared to the condition in which physiological saline was injected, the level of fear emotion was significantly increased (increased freezing behavior, increased heart rate and decreased body temperature) with the # symbol (Student's t-test # : P <0.05, ##: P <0.01, ###: P <0.001). Conversely, compared to the condition in which physiological saline was injected, the level of fear emotion was significantly increased (reduced freezing behavior, reduced heart rate and lower body temperature) with the symbol * (Student's t -test *: P <0.05, **: P <0.01, ***: P <0.001). The difference in memantine and MK801 efficacy could be evaluated by distinguishing even the conditions infused into the amygdala.

Example 12
We analyzed the effects of psychotropic drugs on the decrease in body surface temperature induced by fear odor. A few days before the start of the experiment, the hair on the back of the mouse was removed using a hair removal cream. Psychotropic drugs (diazepam (3 mg / kg), memantine (25 mg / kg), MK801 (0.1 mg / kg), ketamine (5 mg / kg), traxoprodil (30 mg / kg)) or control physiology in mice Saline (Vehicle) was injected intraperitoneally (N = 8). After introducing the mouse into the test cage and acclimatizing, the body surface temperature was measured using a thermography camera under the condition of no odor (No odor) and then the condition of smelling 2MT (2MT).

The results are shown in FIG. In a (smell-free condition) and b (2MT-smelled condition), the case where the body surface temperature is significantly lower than the control is indicated by # symbol (Student's t-test #: P < 0.05, ##: P <0.01, ###: P <0.001). Conversely, the case where the body surface temperature was significantly increased compared to the control was indicated by * (Student's t-test *: P <0.05, **: P <0.01, ***: P <0.001 ). In addition, the difference in body surface temperature under 2MT conditions compared to the condition without odor was calculated (c, 2MT-No odor). The case where the decrease in body surface temperature was significantly reduced is indicated by * (Student's t-test *: P <0.05, **: P <0.01, ***: P <0.001). The efficacy of psychotropic drugs could be classified using the decrease in body surface temperature induced by fear odor as an index.

Example 13
The zodiac sign indicates that the pig takes on the posture of a dog, and is considered a kind of pig's stress response. After sniffing 2MT, pigs were frequently observed to be in the constellation position. On the other hand, under the condition that the smell of spice (eugenol) can be smelled, the kennel was not recognized. Pigs are thought to be stressed by the smell of 2MT.

Example 14
Freezing behavior is a behavior in which rodents such as mice shrug and become stuck, and are thought to be a kind of fear response to prevent them from being discovered by predators, natural enemies. The behavior of pigs when sniffing 245TMT (2,4,5-trimethyl-thiazole) and 2MT (2-methyl-2-thiazoline) was photographed with a video camera. Even in the condition where 245TMT is sniffed, the pig is quiet, but the face and nose are moving. The pig stands up with four legs. On the other hand, under the condition of sniffing 2MT, pigs behave like Freezing and change their face and nose direction little. Also, the pigs are lying down without being able to stand up. 2MT is thought to induce Freezing behavior on this pig. Thus, pigs are thought to be afraid of 2MT.

Example 15
The changes of body temperature to horror odor of pigs were analyzed. The pig body was implanted with a wireless thermometer more than one week before the experiment. The pig is moved from the rearing cage to a moving cage with a lid, and left for 10 minutes under the condition of no odor (white) or under the condition of 2MT (black). (N = 3). 2MT was made to smell by attaching a total of 12 pieces of cotton soaked with 1 ml of 2MT to the front and both sides of the moving cage. The results are shown in FIG. The graph of FIG. 15 shows the mean ± standard error of the body part temperature fluctuation under each condition. *** indicates that the deep body temperature is significantly reduced compared to the odorless condition (Student's t-test ***: P <0.001). 2MT is thought to induce innate horror emotions in pigs, which is measured using a decrease in deep body temperature as an indicator.

Example 16
The change of body surface temperature for horror odor of pigs was analyzed. The body surface temperature was measured by using an infrared thermography camera to measure the temperature of the nose of a pig. The pig was transferred from the rearing cage to a moving cage with a lid and left for 10 minutes under the condition of no odor (white) or under the condition of sniffing 2MT (black), and the body surface temperature at the tip of the nose was measured (N = 3). . 2MT was sniffed in the same manner as in Example 15. The results are shown in FIG. The graph of FIG. 16 shows the mean ± standard error of the body surface temperature of the nose tip. *** indicates that the body surface temperature is significantly increased compared to the odorless condition (Student's t-test ***: P <0.001). 2MT has the effect of significantly increasing the body surface temperature at the tip of the nose. Pigs show fear-related behaviors such as easy-to-seat behavior for 2MT (Examples 13 and 14), and a decrease in deep body temperature that is an indicator of congenital fear (Example 15). Showed a fear response. Therefore, it can be estimated that the pig feels an innate fear of 2MT. In pigs that feel innate fear of 2MT, the body surface temperature of the nose increases, so in pigs, an increase in body surface temperature of the nose may be an indicator of innate fear.

Example 17
Pigs were smelled with spice odor (eugenol), 2MT was sniffed, psychotropic drugs (MK801 or diazepam) were administered by intravenous injection at a dose of 1 mg / kg, and 2MT was sniffed The behavior of pigs was photographed with a video camera under different conditions, and changes in behavior (a) and fluency (b) were analyzed.

The pig behavior was scored 0-2. It was defined that the normal action level was 0, and the higher the score, the more fixed time, that is, more freezing action. Based on the video from the video camera, the behavior of each individual pig was scored. The mean ± standard error of behavioral scores under each condition is shown in the graph of FIG. 17A (N = 6). The symbol * indicates a significant difference between the groups shown in the figure (Student's t-test, *: P <0.05, ***: P <0.001).

The fluent state was scored 0-3. The normal level of fluency was defined as 0, and the state where the amount of fluency was the maximum was defined as 3. A score was assigned to the level of fluency of each pig based on the video camera. The mean ± standard error of the fluency score under each condition is shown in the graph of FIG. The symbol * indicates a significant difference between the groups shown in the figure (Student's t-test, ***: P <0.001).

¡Pigs have a low level of freezing behavior for spicy scents, but if they sniff 2MT, the level of freezing behavior increases. It was also observed that pigs had low fluency levels when they smelled spices, but increased fluency levels when 2MT was smelled. The mechanism by which fear odors promote fluency is unknown at this time, but the amount of fluency can be an indicator of innate fear. Effects of psychotropic drugs were observed on freezing behavior and fluency levels. Based on the level of freezing behavior and fluency, the efficacy of psychotropic drugs could be classified.

Normal fear emotions are necessary in order to trigger alarming and self-protecting actions in situations where dangerous objects or situations are encountered. However, abnormal fear emotions can cause mental illness such as PTSD and anxiety. Since drugs that affect fear emotions may function as psychotropic drugs, a psychotropic drug screening system can be constructed by focusing on fear emotions. Various methods are used to induce fear emotions in experimental animals, and the induction level of fear emotions is measured by methods such as behavioral experiments. At this time, the effect of the psychotropic drug candidate can be analyzed by analyzing the influence of the administration of the psychotropic drug candidate.

恐怖 A variety of sensory inputs can induce congenital and acquired fear emotions. The present inventors screened an artificial odor molecule library, and for the first time, developed an odor molecule group “fear odor” having a much higher fear reaction-inducing activity than secretory components of known carnivores. Developed (WO2011 / 096575). The congenital fear response induced by the fear odor developed here is controlled by a different neural mechanism than the acquired fear response induced by related learning with electric shock that has been analyzed in detail so far It became clear for the first time. Congenital fear reactions due to certain types of fear odors have been unprecedented in mice, including strong freezing behavior, simultaneous 3 ° C decrease in body surface temperature and body temperature, and rapid halving of heart rate. With a typical reaction. In addition, based on the results of analysis using the whole-brain activation mapping method, which is a method of analyzing brain neural activity, congenital fears induced by fear odors are widespread, including the amygdala pathway, striatal pathway, septal pathway, etc. It has been shown that it is controlled by neural activity in the brain region. These specific physiological responses and neural activities cannot be triggered by previously known carnivorous secretions or their congenital or acquired fears. Therefore, by using the fear odor developed by the present inventors, an unprecedented new kind of fear emotion reaction can be induced for the first time. Therefore, it is considered that a new screening method for psychotropic drugs different from the conventional methods can be developed by analyzing the response specificity of a new fear emotion induced by fear odor to a psychotropic drug candidate.

In fact, this technology has developed a new psychotropic drug screening system using the congenital fear emotion reaction induced by fear odor as an index. We analyzed the effects of psychotropic drugs on Freezing behavior and other physiological responses associated with fear emotions induced by fear odor. As a result, it became clear that various physiological responses and freezing behavior associated with congenital fear emotions are affected by different types of psychotropic drugs. Acquired Freezing induced by conventional techniques is less specific because it is affected by various types of psychotropic drugs. In contrast, Freezing behavior induced by certain types of fear odors was not affected by many psychotropic drugs, but some NMDA such as psychotropic drugs and their candidates MK801 and memantine. It was specifically inhibited only by receptor antagonists. Acquired Freezing is controlled by the recall of fear memory and the induction of fear emotions. Therefore, it cannot be distinguished whether the effect of psychotropic drugs on acquired Freezing behavior is on the recall of fear memory or on the fear emotion itself. On the other hand, in the congenital fear due to fear odor, fear emotions are directly induced without going through the memory mechanism, so that the influence of psychotropic drugs on fear emotion itself can be directly analyzed. Therefore, it can be evaluated that the method of the present invention has an advantage that fear emotion can be directly measured.

In addition, by using specific types of fear odors, four or more types of actions including (1) induction of freezing behavior, (2) body surface temperature decrease, (3) body depth temperature decrease, and (4) heart rate decrease, Physiological indicators fluctuate simultaneously, and these indicators show responsiveness to different psychotropic drugs. Therefore, it is considered that various behaviors and physiological responses induced by fear odor are controlled by different molecular targets. With this feature, the method of the present invention makes it possible to carry out screening for molecular targets involved in a plurality of types of emotional responses in one experimental operation of smelling a fear odor. Screening for various targets is possible even with conventional screening technology using behavioral batteries that evaluate the response of psychotropic drugs by behavioral experiments targeting multiple behaviors controlled by different neural mechanisms. is there. While the technique using the behavioral battery needs to perform a plurality of different experiments, the method of the present invention is superior in that it is possible to perform quicker screening in a single experiment.

The method of the present invention is useful as a method for screening a drug for controlling emotion (for example, a preventive or therapeutic drug for mental illness) and a fragrance for inducing a relaxing effect.

This application is based on Japanese Patent Application No. 2012-256514 filed in Japan, the contents of which are incorporated in full herein.

a: soundproof box b: freezing behavior analysis camera c: body surface temperature measurement thermography camera d: rearing cage e: odor molecule generator f: nozzle g: exhaust fan h: illumination i: heartbeat / depth of body temperature measurement receiver j: Heartbeat and body temperature measurement transmitter k: Computer for control and analysis

Claims

(a) a step of administering a test substance to a test animal;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) changing the freezing time, adjusting the decrease in body surface temperature, adjusting the decrease in deep body temperature, or selecting a test substance that adjusts the decrease in heart rate, Screening method for drugs to control
The method according to claim 1, wherein the drug for controlling emotion is a preventive or therapeutic drug for mental illness.
(a) a step of administering a test substance to a test animal;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) measuring the deep body temperature of the test animal,
(d) The method according to claim 1, which is a screening method for a prophylactic or therapeutic agent for psychiatric disorders, comprising the step of selecting a substance that suppresses a decrease in deep body temperature.
The method according to claim 3, wherein the preventive or therapeutic agent for mental illness is a therapeutic agent for intractable depression.
The method according to any one of claims 1 to 4, wherein the test animal is a rodent.
(a) exposing the test animal to the test substance;
(b) exposing the test animal to a substance that induces a congenital fear emotion;
(c) a step of measuring at least one selected from a freezing time, a body surface temperature, a deep body temperature and a heart rate of a test animal,
(d) A step of changing a freezing time, adjusting a decrease in body surface temperature, adjusting a decrease in body depth temperature, or selecting a test substance that regulates a decrease in heart rate. Screening method.
The method according to claim 6, wherein the test animal is a rodent.