WO2003073098A1

WO2003073098A1 - Method of screening psychotropic

Info

Publication number: WO2003073098A1
Application number: PCT/JP2003/002367
Authority: WO
Inventors: Noriko Osumi; Kaoru Inokuchi; Michio Fujiwara
Original assignee: Japan Science And Technology Agency; Yamanouchi Pharmaceutical Co., Ltd.; Mitsubishi Chemical Corporation
Priority date: 2002-02-28
Filing date: 2003-02-28
Publication date: 2003-09-04
Also published as: JPWO2003073098A1; JP4106030B2

Abstract

It is intended to provide an animal model appropriately corresponding to human schizophrenia, a method of screening a psychotropic with the use of the animal model, etc. A hetero rat with Pax-6 mutation, which is constructed by deleting Pax6 gene exclusively in one of allele locus and thus shows behavioral disorders similar to the symptom of human schizophrenia, is employed as a schizophrenia model animal. A psychotropic is screened by administering a test substance to the model animal and measuring/evaluating changes in the behavioral disorders similar to the symptom of human schizophrenia. The behavioral disorders exhibited by the model animal include behavioral disorders with worsened sensory gate mechanism, behavioral disorders with insufficient memory retention due to fear, behavioral disorders with lack of sociality, behavioral disorders with loss in fear and behavioral disorders with loss in curiosity, compared with the wild type.

Description

Description Screening method for antipsychotic drugs Technical field

INDUSTRIAL APPLICABILITY The present invention is used for a screening method of an antipsychotic drug using a non-human animal having a mutation in the PaX6 gene and exhibiting a behavioral abnormality serving as an indicator of a human mental illness, or a screening method thereof For animals and so on. Background art

The P aX gene has a homeobox and a paired box and is a member of the homeobox gene encoding a transcription factor, which is classified into 1 to 9 subfamilies. It is known to be involved in eye morphogenesis and nervous system development in a wide variety of species, from insects to mammals. In order to elucidate the function of this homeobox-type transcription factor called PaX6 (for example, see Non-patent Documents 1 to 5) in brain neurogenesis, the present inventors have proposed a method in which only one allele locus is used. Analysis using a rat having a mutation that introduces a base into the gene encoding ax6 (Small eye rat: r Sey2, hereinafter sometimes referred to as “Pax6 mutant heterorat”) PaX6 is expressed in fetal brain, eyes, and kidney, etc., and homozygous individuals with FaX6 mutation heterozygotes show severe developmental abnormalities in these organs. Is revealed. That is, abnormalities such as cerebral neocortical layer formation, olfactory bulb formation, thalamocortical tract formation, cerebellar formation, neuronal and Dalya differentiation of the rhombic and spinal cords have already been reported (for example, see Non-Patent Documents 6 to 11). ).

P ax 6 gene is important in memory and emotional function in hippocampus and tonsil in postnatal brain Homozygous individuals that are expressed in the body and have a Pax6 mutant rat die shortly after birth, but heterozygous individuals do not show any noticeable developmental abnormalities other than microphthalmia, etc. Can be.

Even in mice, there are individuals spontaneously having mutations in the PaX6 gene (for example, see Non-Patent Documents 12 to 14). The mutation in the PaX6 gene of this individual has a base inserted 3 'downstream, and the mutation in the Pax6 gene expressed by this mutation has several amino acids at the C-terminus. It has been added. This mutant also shows a phenotype similar to that of the PaX6 heterolat. Furthermore, humans have been reported to have a mutation in PaX6. Among them, it has been reported that individuals with mutations at any of the 100th, 1336th, or 240th amino acid numbers cause a genetic disease called aniridia ( For example, see Non-Patent Document 15).

On the other hand, as an individual with a human PaX6 mutation, the degree of promoter activation of Pax6 with more than 29 ACZAG repeats in the 5 'transcriptional control region of PaX6 is high. A mild association with paranoid schizophrenia has been suggested in a population (eg, see Non-Patent Document 16).

Considering the relationship between these P aX6 mutants and their phenotypes, it is clear that mutations in the Pa6 gene show some phenotypic change, all of which are This suggests that PaX6 is functioning. However, as described above, since the positions and types of mutations of PaX6 possessed by each mutant are different, it is not possible to universally predict the mutation of the PaX6 gene and its phenotype from these findings. Have difficulty.

In animals with mutations in the Pax6 gene, including the Pa6 mutation heterolat, the neurogenesis was analyzed, but the behavioral abnormalities were not analyzed. . The animal In the behavior of the subject, in particular, if there is an abnormality associated with a human neurological disease, the animal can be used as a model animal for the disease, but as described above, the mutation of the P aX6 gene Since it is difficult to predict abnormalities in the behavior from the data, it is desired to analyze the behavior. Non-Patent Document 1 Cell, 69, 719-722, 1992

Non-Patent Document 2 Curr.Opin.Cell Biol., 4, 967-972, 1992

Non-Patent Document 3 Special Issue on Experimental Medicine 14 (8), 186-192, 1996

Non-patent document 4 Cell engineering 18, 1838-1845, 1999

Non-Patent Document 5 Tohoku J. Exp.Med., 193, 163-180, 2001

Non-Patent Document 6 Nature Genet., 3, 299-304, 1993

Non-Patent Document 7 Differentiation, 57, 31-38, 1994

Non-Patent Document 8 Development, 124, 2961-2967, 1997

Non-Patent Document 9 J. Comp.Neurol., 408, 147-60, 1999

Non-Patent Document 10 Dev. Brain Res., 120, 65-75, 2000

Non-Patent Document 1 1 Development, 128, 3133-3144, 2001

Non-Patent Document 1 2 Nature, 354, 522-525, 1991

Non-patent document 13 J.Embryol.Exp.Morph., 97, 95-110, 1986 Non-patent document 14 Acta.Neuropathol., 86, 126-135, 1993

Non-Patent Document 15 Trendsin Genetics, 11, 68-272, 1995

Non-Patent Document 16 Gerald, S, Biol. Psychiatry, 45, 1585-1591 1999 Schizophrenia (schizophrenia) is a functional psychiatric disorder mainly due to a predisposing factor, and often involves a genetic predisposition It has been. Vulnerability to stress has become an issue as a basis for the pathogenesis of schizophrenia, and schizophrenia patients are on the rise in today's high-stress society. Schizophrenia The elucidation of the basis for the onset of the disease has been limited in terms of methods, and the identification of the causative gene and the creation of a model animal with a similar disease state and course have not yet been achieved. In order to develop effective antipsychotic drugs, it is necessary to first determine the effects of the drug in an appropriate animal model for human schizophrenia. However, the model animals provided to date have been administered stimulants or narcotics, and no appropriate animal models have been provided. An object of the present invention is to provide an appropriate animal model corresponding to a human psychiatric disorder, a method for screening an antipsychotic drug using such an animal model, and the like.

The present inventors were aware that this rat showed reduced fear while maintaining the lineage of the PaX6 mutant heterolat, and therefore analyzed the behavior of the Pax6 mutant heterolat. Was done. As a result, they found that the Pax6 mutation heterozygous exhibited behavioral abnormalities that are indicative of human mental illness. In other words, the P aX6 mutation heterolatate is remarkable in social tests, light and dark tests, fear conditioning tests, prepulse suppression tests, etc., although there is no difference in wild-type in general activity. Significant differences were noted, indicating reduced curiosity and fear, lack of sociality, and decreased sensory-gating mechanisms.

The strain of the Pa 6 mutant heterolat has a homogeneous genetic background, a large number of offspring, high fertility, and symptoms similar to human schizophrenia without drug administration. It was considered to be an appropriate animal model for human schizophrenia and was found to be extremely useful as an experimental animal for the development of antipsychotic drugs. The present invention has been completed based on these findings. Disclosure of the invention

That is, the present invention relates to a method for administering a test substance to a non-human animal having a mutation in the PaX6 gene and exhibiting a behavioral abnormality that is an indicator of a human psychiatric disorder, The screening method for an antipsychotic drug, which is characterized in that a substance is selected by using the change of the index as an index (Claim 1), and wherein the behavioral abnormality is a decrease in a sensory gate mechanism. An antipsychotic screening method (claim 2), an antipsychotic screening method according to claim 2, wherein the antipsychotic is a treatment for schizophrenia (claim 3), The method for screening an antipsychotic drug according to claim 1, wherein the behavioral abnormality is a decrease in fear conditioning ability, a lack of sociality, a decrease in fear, or a decrease in curiosity (claim 4), and the P aX6 gene. A drug for treating schizophrenia, which comprises administering a test substance to a non-human animal having a mutation in the sensory gate and exhibiting a decrease in the sensory gate mechanism, and selecting a substance using the sensory gate mechanism of the animal as an index. Screening method (Claim 5) ) Or the animal is a mouse or a rat. The screening method according to any one of claims 1 to 5 (claim 6), and an animal for use in the screening method according to claim 1 to 6 (claim 7) A method for formulating a drug for a psychiatric psychiatric drug, which comprises formulating a substance selected by the screening method according to claims 1 to 6 (claim 8). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a general activity test of a PaX6 mutant heterolat and a wild type rat.

FIG. 2 is a diagram showing the results of a light-dark test of a PaX6 mutant heterolat and a wild-type rat.

FIG. 3 shows the results of a social test of the Pax6 mutant heterolat and wild-type rat.

FIG. 4 is a diagram showing the results of a fear conditioning test of the Pax6 mutant heterolat and wild-type rats.

Figure 5 shows the prepulse suppression of the PaX6 mutant heterozygous rat and the wild-type rat. It is a figure showing a result of a control test.

FIG. 6 is a graph showing the results of a prepulse suppression test for the sound stimulus of the Pax6 mutant heterorat administered with clozapine.

FIG. 7 shows the results of a fear conditioning test using the sound of a Pax6 heterolat administered with clozapine.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Mental disease model animal

The animal used in the present invention includes an animal having a mutation in the P aX6 gene in its chromosome and exhibiting a behavioral abnormality that is an indicator of a human mental illness (hereinafter referred to as a “mental illness model animal”). Is not particularly limited. Any animal species may be used as long as it has the PaX6 gene in the chromosome.For example, rodents such as mice and rats, egrets, dogs, dogs, A lodge, a monkey and the like. In the present invention, the Pax6 gene refers to not only the translation region but also all regions related to the expression of the PaX6 protein. Specifically, for example, the rat Pax gene includes the gene shown in Genbank accession NO.III-013001, and the mouse PaX6 gene includes Walther, C. et al., Genomics, 11 ( 2), 424-434 (1991), but are not limited thereto.A DNA fragment having the above nucleotide sequence in the chromosome of the animal used in the present invention and comprising Means the area obtained by the hybridization etc. Here, when the expression control region of the Pax6 gene is obtained, it is confirmed whether or not the obtained DNA fragment has the activity of controlling the expression of PaX6 by a method known per se and generally used. I can do it. Specifically, for example, a suitable marker 3 ′ downstream of the DNA fragment After binding each gene and, if necessary, binding a termination region or the like, introducing this into an appropriate cell or the like, and confirming the expression of the marker gene in the cell, etc. Is mentioned.

Having a mutation in the PaX6 gene means that the nucleotide sequence of the wild-type PaX6 gene of the animal used has changed by one or more bases. Specific examples of the mutation include substitution, deletion, and insertion of DNA. These mutations may be spontaneous or introduced artificially. The fact that the Pax6 gene has a mutation can be confirmed by a commonly used method known per se. Specifically, for example, a cDNA or a genomic library is prepared from appropriate cells of the animal, and DNA containing the Pax6 gene is simply isolated from the library by using polymerase chain reaction (PCR) or the like. And comparing the nucleotide sequence with the wild type.

As a method for artificially introducing a gene mutation, a commonly used method known per se can be used. For example, a knockout method (the latest technology of gene targeting, Ken Yagi, Yodosha, 2 Nokiin method (the latest technology of gene targeting, Ken Yagi, Yodosha, 2000), Transgenic method (Transgenic animal, Kenichi Yamamura et al., Edited by Kyoritsu Shuppan, 1992) 7 years). The mutation of the PaX6 gene may be a mutation in either one or both of the two loci on the homologous chromosome of the animal used. Here, an animal having a mutation in one of the P aX6 genes at one of the alleles (hereinafter, this mutation may be referred to as “heterotype”) is obtained from both animals (hereinafter, this mutation is referred to as “heterotype”). As a method for obtaining an animal having the homozygous form, the male and female of the animal having the heterozygous mutation are bred, and the genotype of the resulting offspring is analyzed to obtain a homozygous animal. To select those with mutations Method.

In addition, a gene having a mutation in a gene other than the PaX6 gene can be used in the present invention as long as it exhibits the following behavioral abnormality.

The animal used in the present invention has a mutation in the PaX6 gene as described above, and further exhibits a behavioral abnormality that is an indicator of human mental illness. Behavioral abnormalities that are indicators of human mental illness include, for example, behavioral decline in sensory gate mechanisms. Behavioral abnormalities, behavioral abnormalities with poor memory due to fear, behavioral abnormalities lacking sociality, and reduced fear. Behavioral abnormalities, or behavioral abnormalities that reduce curiosity, and the like. Of these, behavioral abnormalities in which the sensory gate mechanism is particularly reduced are preferable because their association with schizophrenia has been elucidated (Braff, et al., Psychopharmacology, 156, 234-258 (2001)> Example 5).

As a method for analyzing and identifying these behavioral abnormalities, a method known per se can be used. Specifically, for example, a method for analyzing the sensory gate mechanism includes a prepulse suppression test by sound stimulation (Braff, et al., Psychopharmacology, 156, 234-258 (2001)). The sensory gate mechanism means that when a slightly smaller stimulus (prepulse) is given just before a large stimulus, the degree of reflex to the stimulus is smaller than that not given. The method of this test is as follows. For example, put the target animal in the measuring device, first listen to the prepulse sound (68, 71 or 77 db) for 20 msec, and then After 100 ms, the stimulus sound (105 db) is heard for 40 ms. At this time, the acceleration of the animal in response to the sound is measured. If this value is larger than that of the wild type, it can be determined that the sensory gate mechanism has decreased, and the animal can be used as a neurological disease model animal of the present invention, preferably a schizophrenia model animal.

Behavioral abnormalities with insufficient conditioning due to fear described above include, for example, fear due to sound. It can be observed and identified by the fear conditioning test. One male rat was placed in the observation box for sound conditioning, and after 2 minutes, the sound was heard for 20 seconds, and immediately after that, an electric shock of 0.3 mA was applied for 1 second to give an electric shock. After 5 minutes, transfer to a rearing cage, and after a predetermined time from the conditioning, put one male rat in the sound conditioning fear test box, and after 2 minutes, listen to the same sound used for conditioning for 5 minutes, and condition The time spent in freezing reflex due to fear was measured, and if the time spent in freezing reflex decreased compared to the wild type, it can be judged that the conditioning by fear was insufficient.

Behavioral abnormalities lacking sociality can be observed and identified by sociality tests. For example, put two male rats kept separately in an open field box, record their behavior on videotape for a predetermined time, and then record the total activity time and its contents (sniff the opponent, Analyze the trailing, grooming, jumping on, fighting, etc.) and reduce the opponent's trailing action (Foll owi ng) compared to the wild type. Conversely, fighting (fighting) If Aggresion is increasing, it can be determined that the behavioral abnormality lacks sociality.

The above behavioral anomalies that reduce fear can be observed and identified by, for example, a light / dark selection test. Rats usually feel scared in bright places and tend to prefer dark rooms. One male rat was placed in the light room of the light / dark field box, and the number of times that it passed through the partition from the dark room and the time spent in the light room were recorded for a predetermined time using a photosensor, and the light was compared with that of the wild type. When both the number of times of movement between the room and the dark room and the time spent in the bright room show large values, it is possible to judge that the behavior is abnormal because the fear is reduced even in the bright room.

Behavioral abnormalities that reduce curiosity include, for example, general activity tests Observe and identify. Put one male rat in the open field box, record the general activity for a predetermined time using a photo sensor, and then record the time spent during the activity, the distance traveled, and the rising behavior (indicating curiosity) When there was no difference in activity time, travel distance, and travel speed compared to the wild type, and when the rising action time indicating curiosity was less than that in the wild type However, it is possible to judge that the behavior is abnormal in which curiosity decreases.

Examples of such a neurological disease model animal include a rat having a mutation in which cytosine (C) is inserted at the 777th position of the Pa6 gene (Development, 128, 3133-3). 144, 200 1) and those that show abnormal behaviors such as a decrease in sensory gate mechanism, a decrease in fear conditioning ability, a lack of sociality, a decrease in fear, or a decrease in curiosity. It is clear that the above rat is particularly inferior in the sensory gate mechanism as compared with the wild type, and thus it is preferable to use it as a model animal for schizophrenia.

(2) Screening method of antipsychotic drug using psychiatric disease model animal The psychiatric disease model animal of the present invention is used as a model animal for elucidating psychiatric disorders including schizophrenia and its pathology at a molecular level, or It is extremely useful as a model animal for the development of therapeutics for these diseases and conditions. By using such a model animal for psychiatric disorders and comparing it with a wild-type rat, PaX6 and its downstream factors Screening for antipsychotic drugs targeting Here, antipsychotics refer to drugs that are effective in quenching excitement such as hallucinations and mania in mental disorders such as schizophrenia. It is characterized by calming emotions without lowering the level of consciousness and reducing interest in the surroundings.

As a method for screening an antipsychotic drug of the present invention, a test substance is administered to a psychiatric disease model animal of the present invention, and the model animal is an indicator of a human psychiatric disorder.

0 The method is not particularly limited as long as it is a method for measuring and evaluating changes in behavioral abnormalities. Of these, changes in behavioral abnormalities that are indicators of human schizophrenia include changes in the degree of disappearance and improvement of behavioral abnormalities with inferior sensory gate mechanisms. In addition, disappearance of behavioral abnormalities that are insufficiently memorized due to fearChange in the degree of improvement, disappearance of behavioral abnormalities lacking socialityChange in the degree of improvement, disappearance of behavioral abnormalities that reduce fear, and degree of improvement And behavioral abnormalities that reduce curiosity and changes in the degree of improvement can also be specifically exemplified as behavioral abnormalities that are indicators of human mental illness. In the screening, the degree of change in disappearance and improvement of behavioral abnormalities, which are indicators of a human psychiatric disorder, in the psychiatric disease model animal of the present invention, a wild-type non-human animal, preferably a litter-like wild-type non-human It is preferable to compare and evaluate the disappearance of similar behavioral abnormalities in animals and the degree of change in improvement.

By using the antipsychotic screening method of the present invention, it is possible to screen therapeutic agents for psychiatric diseases targeting PaX6 and its downstream factors. The method of formulating the antipsychotic drug of the present invention may be any method as long as it is a method of formulating a substance selected by the screening method of the antipsychotic drug. When used as pharmaceuticals, ordinary pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents And other commonly used dosage forms such as powders, granules, capsules, syrups, suspensions, etc., or For example, solutions, emulsions, suspensions and the like can be administered parenterally in the form of injections.

By administering the therapeutic agent of the present invention to an individual (patient) who is likely to have a mental disease, the disease can be treated. Those skilled in the art will be able to determine the appropriate treatment according to the administration route, dosage form, type of administration subject, age, body weight, etc. of the therapeutic drug of the present invention. It is easy to select the administration timing and dosage. Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.

(PaX6 mutation heterolate)

Heterozygous rat having a mutation in a gene encoding a home box transcription factor called P aX6 (Smal 1 eye rat; rSey2 Matsuo T, et al., Nature Genet., 3, 299-304, 1993; Osumi Ν Development, 124, 2961-2967, 1997; hereinafter, sometimes referred to as “Pax 6 mutant heterolatato”, 8 to 16 weeks old, and a wild-type SD rat of the same litter Sometimes referred to as “wild type”). This mutation in the PaX6 mutant heterorat has a cytosine (C) inserted at the 777th position of the Pax6 gene. The stop codon appears at the fourth and third place from the translation start point.

(Behavior analysis device)

Open field box: An acrylic box measuring 80 x 80 x 40 cm with 24 x 24 light beam sensors mounted at 2 cm and 14 cm height from the bottom, Used for general activity tests and social tests. The measurement was performed in a silent room, and the input from the light beam sensor was recorded on a computer, and the behavior was recorded on videotape.

Light and dark field box: A box the same size as the open field box above is divided into two, and one is illuminated with an incandescent bulb to make it bright (2 50 lux), one is dark (1 lux). The wall that separates the two rooms has an opening of 1 2 x 25 cm, through which the light beam sensor runs. The test was performed in a dark room with no sound.

Observation box for sound conditioning (Muromachi Kikai, custom-made): H2 8 XD 24 XW30 Cm box is divided into triangles by transparent and gray plastic panels. <The floor of the box is made of 19 stainless steel. A rod has been handed over and can give an electric shock. Sound conditioning was performed in a silent room.

Fear test box with sound conditioning (Muromachi Kikai, custom-ordered product): A box (W3 2 XD 2 2 XH 2 2 cm, square) with a shape different from the observation box for sound conditioning. The fear was measured by freezing reflex, and the fear was measured in a silent room.

Pre-pulse suppression test device (SR-Lab systems, San Diego Instruments): A device that puts a rat in a cylindrical container and measures startle with a pressure sensor. The measurement of the prepulse suppression test by sound stimulation was performed in a silent room.

Example 1 (General activity test)

Twenty-four (24) wild-type and thirty-four (4) PaX6 mutant heterolatants were analyzed for their behavior in an open field box for 30 minutes. Each rat was placed in an open field box, and general activity was recorded for 30 minutes using a photosensor. By analyzing these results, we calculated the time spent in activity, the distance traveled, and the time of standing up (indicating curiosity). As a result, there was no difference between the wild type and the model rat of the present invention in terms of the time spent in activity (Fig. 1A), the distance traveled (Fig. 1B), and the travel speed (Fig. 1C). With regard to standing behavior (Rearing; Fig. 1D), the number of Pax 6 mutant heterolatants was less than that of wild type. In other words, the Pax 6 mutant heterolatate tended to show no interest in the new environment.

Example 2 (Light / dark selection test)

Twenty-four (24) wild-type and thirty-four (24) PaX6 mutant heterolats were measured in the light-dark field box for 30 minutes. The rats were placed one by one in the light room of the light-dark field box, and the number of passages through the partition from the dark room and the time spent in the light room were recorded for 30 minutes using a photosensor. As a result, both the number of transitions between the light room and the dark room (Transition; Fig. 2A) and the percentage of time spent in the light room (Fig. 2B) were larger in the PaX6 mutant heterolatate than in the wild type. showed that. In other words, it was found that the wild type rat tended to fear in the light room and prefer the dark room, whereas the PaX6 mutant heterolat did not feel much fear in the light room.

Example 3 (Social test)

For 24 wild-type and 34 PaX6 mutant heterolats, two animals each were placed in an open field box, and the behavior recorded on videotape was analyzed. Two male rats of the same species, which were kept separately, were placed in an open field box, and their behavior was recorded on a videotape for 15 minutes. This was analyzed, and the total time spent during the activity and its contents (sniffing, smuggling, grooming, riding, fighting, etc.) were analyzed. As a result, there was no significant difference in the behavior of sniffing, sniffing, grooming, and riding (Mounting), but Pax 6 mutant heterozygous. In the results, follow-up behavior (Following) was about 40% less, and quarrel-fighting behavior (Aggression) increased to more than 300% (Fig. 3). It was suggested that heterorats tended to lack sociality. Example 4 (Sound fear conditioning test)

Fear conditioning by sound was performed on 26 wild-type cases and 36 Pa6 mutant heterolats. The rats were placed one by one in the observation box for sound conditioning, and after 2 minutes, the sound was heard for 20 seconds (Tone). Immediately after that, an electric shock of 0.3 mA was applied for 1 second to give an electric shock. Was. After 5 minutes, transfer to the rearing cage, 48 hours and 96 hours after conditioning, re-sound the sound, put it in the fear test box, and after 2 minutes, listen to the same sound as used for conditioning for 5 minutes. The time of freezing reflection was measured by the fear given. The time of freezing reflex was measured by the number of seconds during which 60 seconds had not passed, and this was shown as a percentage. The results at 48 hours (FIG. 4A) and at 96 hours (FIG. 4B) indicated that fear conditioning was not sufficient in the PaX6 mutant heterolat. For two wild-type and two PaX6 mutant heterolats, only sound conditioning (Tone) was performed, and if no fear conditioning was performed, freezing reflex occurred 48 hours after sound conditioning. Time was hardly observed in both cases. Example 5 (Prepulse suppression test by sound stimulation)

A prepulse suppression test by sound stimulation was performed on 21 wild-type cases and 36 Pa6 mutant heterolats. Each rat was placed in a measuring device one by one, and the startle response when only the main stimulus (105 dB, 40 ms) was heard was measured (Fig. 5A). The startle response was measured as the magnitude of the acceleration of the backward response after the rat. Next, to measure the pre-pulse suppression due to sound, give a pre-pulse sound (68, 71 or 77 dB) of 100 ms before 100 ms of this stimulus sound.

The startle response to the main stimulus sound (105 dB) was measured (Fig. 5A). Prepulse suppression was expressed as 11 S pp / S (S pp: startle response to main stimulus when prepulse was heard, S: only main stimulus sound Startle reaction when). As a result, the rate of prepulse suppression was significantly lower for the 68-dB prepulse sound in the P aX6 heterolatate than in the wild type (P test, P <0.01, soft name: S tarview) (Fig. 5B). Therefore, it was found that the sensory gate mechanism of the PaX6 hetero rat was lower than that of the wild type.

Example 6

(1) (Test for suppression of prepulse to sound stimulation of PaX6 mutant heterolat administered clozapine)

Clozapine (1.5 mg / kg), which is used as an antipsychotic for the treatment of schizophrenia, was injected intraperitoneally into 11 wild-type and 11 Pa6 mutant heterolats. Was administered. As a control, the same amount of physiological saline was injected intraperitoneally into 11 wild-type and 11 PaX6 mutant heterolats. A prepulse suppression test by sound stimulation was performed on the above rats 30 minutes to 2 hours after administration. Put the above rats one by one into the measuring device, and first, before 100 msec before the main stimulus sound (120 dB, 40 msec), pre-pulse sound of 20 msec (68, 71) Alternatively, the startle response to the main stimulus tone (120 dB) when listening to 77 dB) was measured. The startle response was measured as the magnitude of the acceleration of the rat's backward reaction. The prepulse suppression was expressed as 11 Spp / S (Spp: startle response to the main stimulus sound when the prepulse was heard, and S: startle response when only the main stimulus sound was heard).

Figure 6 shows the results. In the figure, the black bar shows the results of the animals to which clozapine was administered, and the white bar shows the results of the control animals. As is evident from the figure, the symptoms with a significantly lower prepulse suppression rate compared to the wild type and the 68, 7 ldB prepulse observed with the PaX6 heterolatate were not affected by clozapine administration. Clearly improved. Also, such a change

6 It was not found in raw rats.

(2) (Test of fear condition by sound of PaX6 heterolat administered clozapine)

Clozapine (1.5 mg / kg), which is used as an antipsychotic drug for the treatment of schizophrenia, was injected intraperitoneally into 6 wild-type and 6 PaX6 mutant heterolats . As a control, the same amount of physiological saline was injected intraperitoneally into 6 wild-type and 6 Pa6 mutant heterolats. A fear conditioning test by sound was performed on the rats 30 minutes to 2 hours after administration. The above rats were placed one by one in the observation box for sound conditioning, and after 2 minutes, the sound was heard for 20 seconds (Tone). Shocked. After 5 minutes, transfer to the rearing cage, 48 hours after conditioning, put it in the sound conditioning fear test box again, and after 1 minute, listen to the same sound as used for conditioning for 6 minutes, then free up according to the conditioned fear The time of reflection was measured. The number of seconds during which the free reflex was not moving was measured, and this was shown as a percentage.

The result is shown in FIG. In the figure, the graph indicated by 鲁 indicates the result of the animal to which clozapine was administered, and the graph indicated by 〇 indicates the result of the control animal. As is clear from the figure, the symptoms of poor fear conditioning seen in the PaX6 mutant heterolat were not improved by clozapine administration.

By administering clozapine to the Pax6 mutant heterolat of the present invention, of the behavioral abnormalities, only the decrease in the sensory gate mechanism was improved. This indicates that a substance having high selectivity for schizophrenia can be selected by using the change in the sensory gate mechanism of the animal as an index.

7 Industrial applicability

Since the psychiatric disease model animal of the present invention exhibits a behavioral abnormality that is an indicator of a psychiatric disorder such as schizophrenia without administration of a drug, an appropriate animal model corresponding to a psychiatric disorder such as human schizophrenia can be obtained. Therefore, it is extremely useful as a model animal for developing new antipsychotics. In particular, strains of the Pa6 mutant heterolat have a uniform genetic background, have more litters, have higher fertility, and do not receive drugs than strains of mice carrying the same gene mutation. Since it exhibits behavioral abnormalities that are indicators of mental illness such as human schizophrenia in a state, it is an appropriate animal model for mental illness such as human schizophrenia.

Claims

The scope of the claims

1. Administer a test substance to a non-human animal having a mutation in the Pax6 gene and exhibiting a behavioral abnormality that is an indicator of a human mental illness, and selecting a substance using the change in the behavioral abnormality as an index. Screening method of antipsychotic drug characterized by <

2. The method for screening an antipsychotic according to claim 1, wherein the behavioral abnormality is a decrease in a sensory gate mechanism.

3. The method for screening an antipsychotic according to claim 2, wherein the antipsychotic is a therapeutic agent for schizophrenia.

4. The screening method for an antipsychotic according to claim 1, wherein the behavioral abnormality is a decrease in fear conditioning ability, a lack of sociality, a decrease in fear, or a decrease in curiosity.

5. A test substance is administered to a non-human animal having a mutation in the PaX6 gene and showing a decrease in the sensory gate mechanism, and a substance is selected using the sensory gate mechanism of the animal as an index. Screening methods for anti-schizophrenia drugs,

6. The screening method according to any one of claims 1 to 5, wherein the animal is a mouse or a rat.

7. An animal for use in the screening method according to claims 1 to 6.

8. A method for formulating an antipsychotic drug, which comprises formulating a substance selected by the screening method according to claims 1 to 6.

9