WO2022138943A1 - Production method for non-human model animal with mental disorder or neurological disorder, said non-human model animal, and method for screening for prophylactic or therapeutic agent for said mental disorder or neurological disorder - Google Patents

Abstract

Provided are: a method for producing a non-human model animal with a mental disorder or neurological disorder that is attributed to an increase or decrease in the expression of an AMPA receptor gene or an increase or decrease in the expression or function of an AMPA receptor in a predetermined region in the brain; a non-human model animal with the mental disorder or neurological disorder; and a screening method for discovering a prophylactic or therapeutic agent for the mental disorder or neurological disorder, using the non-human model animal.　This method is for producing a non-human model animal with a mental disorder or neurological disorder that is attributed to an increase or decrease in the expression of an AMPA receptor gene or an increase or decrease in the expression or function of an AMPA receptor in a predetermined region in the brain. The method comprises: a step for decreasing or increasing at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor, and the function of the AMPA receptor, in the predetermined region of the non-human animal, compared to a wild type, or a step for inhibiting or activating a nervous pathway connecting the predetermined region with another region.

Description

A method for producing a model non-human animal for a psychiatric disorder or a neurological disorder, a method for screening the model non-human animal, and a prophylactic or therapeutic agent for the psychiatric disorder or the neurological disorder.

The present invention relates to a method for producing a model non-human animal for a psychiatric disorder or a neurological disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. The present invention relates to a non-human animal model of a psychiatric disorder or a neurological disorder, and a screening method for discovering a prophylactic or therapeutic agent for the psychiatric disorder or a neurological disorder using the model non-human animal.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptor is a synaptic functional molecule and plays an important role in neuropsychiatric activity. The present inventors have clarified that AMPA receptor (glutamate receptor) synaptic translocation in vivo is a molecular cell mechanism of plastic phenomena of various higher brain functions such as memory learning (for example). , Non-Patent Documents 1-8). In recent years, it has been reported that AMPA receptors are involved in many psychiatric and neurological disorders such as bipolar disorder, schizophrenia, drug dependence, autism, epilepsy, and ALS, mainly in the field of postmortem brain research. Has been done. Preclinical and clinical development of AMPA receptor agonists is being actively carried out for such diseases.

The present inventors have obtained a patent for a novel compound that specifically binds to an AMPA receptor (see Patent Document 1), and in Non-Patent Document 9, in animals and humans (healthy subjects, epilepsy patients), the above compounds are used. Shows effectiveness. In addition, a patent application has been filed for an imaging method of AMPA receptors in the brain of a primate organism (see Patent Document 2).

Japanese Patent No. 6425817 International release WO2018 / 131663

As a result of diligent studies based on the above techniques, the present inventors have found that human psychiatric disorders or neurological disorders have increased or decreased expression of AMPA receptor gene in a predetermined region in the brain, or expression or function of AMPA receptor. We find that it correlates with an increase or decrease. Specifically, as the above correlation, for example, the following (1) to (5) are found.
(1) In the manic state based on the YMRS rating scale of human bipolar disorder patients, the AMPA receptor density in the cerebellum decreases, that is, there is a negative correlation.
(2) In depression based on the HAM-D rating scale of human bipolar disorder patients, there is an increase in AMPA receptor density in the parietal lobe and occipital lobe, that is, there is a positive correlation.
(3) In human schizophrenia patients, AMPA receptor density is relatively low in the cingulate gyrus.
(4) In human autism patients, AMPA receptor density increases in the middle frontal gyrus orbit, and AMPA receptor density decreases in the occipital lobe and cerebellum.
(5) In human depression patients, AMPA receptor density decreases in the frontal lobe and cerebellum.

The present invention has been made in view of the above findings, and is a psychiatric disorder or neurological disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. A method for producing a disease model non-human animal, a model non-human animal for the psychiatric disorder or a neurological disorder, and a screening for creating a prophylactic or therapeutic agent for the psychiatric disorder or the neurological disorder using the model non-human animal. The purpose is to provide a method of doing so.

The present inventors knock down the expression of the AMPA receptor gene in a predetermined region in the brain of a mouse or rat, or increase the function of the AMPA receptor, or the above-mentioned predetermined region and others. It has been further found that the mouse or rat exhibits a corresponding mental or neurological disorder by suppressing or activating a neural pathway connecting the region (region other than the predetermined region). More specifically, for example, the following (6) to (11) have been further found.
(6) By knocking down the AMPA receptor gene in the cerebellum of a mouse, the mouse significantly exhibits a manic state similar to that of a patient with bipolar disorder.
(7) By knocking down the AMPA receptor gene during the anterior cingulate gyrus of the mouse, the mouse significantly exhibits a schizophrenic state.
(8) By increasing the AMPA receptor in the middle frontal gyrus orbit of a mouse, the mouse significantly exhibits an autistic state.
(9) In a mouse, by suppressing the claustrum having a neural connection with the anterior cingulate cortex and the neural connection with the anterior cingulate cortex, the mouse significantly exhibits a schizophrenic state.
(10) By knocking down the AMPA receptor gene in the medial prefrontal cortex of a rat, the rat significantly exhibits a depressive state.
(11) In rats, by suppressing the neural connection between the basolateral amygdala nucleus, which has a neural connection with the medial prefrontal cortex, and the anterior cingulate cortex, the rat significantly exhibits a depressive state.
The present invention has been completed based on the above findings.
Specifically, the present invention is as follows.

The first aspect of the present invention is a model non-human animal of a mental disease or a neurological disease caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. It is a manufacturing method of
A step of reducing or increasing (improving) at least one selected from the group consisting of the expression of an AMPA receptor gene or AMPA receptor in the predetermined region of a non-human animal and the function of the AMPA receptor as compared with the wild type. , Or a method comprising a step of suppressing or activating a neural pathway (nerve transmission pathway) connecting the predetermined region and another region (brain region other than the predetermined region).
A second aspect of the invention is a model non-human animal for mental or neurological disorders resulting from increased or decreased expression of the AMPA receptor gene in a predetermined region of the brain or increased or decreased expression or function of the AMPA receptor. And
At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor is decreased or increased (improved) as compared with the wild type, or the predetermined region. A model non-human animal in which the neural pathways connecting one region to another are suppressed or activated.
A third aspect of the present invention is a method for screening a prophylactic or therapeutic agent for a psychiatric disorder or a neurological disease, wherein the test substance is administered to the animal according to the second aspect, and the psychiatric disorder or the neurological disease in the animal is concerned. It is a method of screening a prophylactic or therapeutic agent for the psychiatric disorder or a neurological disorder by using the prevention, suppression or cure of the above as an index.

The model non-human animal of the psychiatric disorder or the neurological disorder according to the second aspect is a psychiatric disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. It is useful as a model animal for a disease or neurological disorder (eg, at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism). The production method according to the first aspect can produce a model animal for the above-mentioned mental disease or neurological disease.
The screening method according to the third aspect can screen a prophylactic or therapeutic agent for a psychiatric disorder or a neurological disorder.

6 is a brain atlas showing the coordinates of introduction of shRNA into the cerebellum of mice for AMPA receptors. It is a figure which shows the result of the posalt forced swimming test. It is a figure which shows the tail suspension test result. It is a synthetic figure of the brain atlas which shows the introduction coordinates of the shRNA for the AMPA receptor into the anterior cingulate gyrus of the mouse, and the brain section GFP fluorescence imaging result for confirmation of the introduction position. It is a figure which shows the prepulse inhibition test result. It is a figure which shows the open field test result. (A) is a diagram showing the results of a three-chamber social interaction (social preference) test, and (b) is a diagram showing a test device. It is a composite diagram of the brain atlas showing the introduction coordinates to the middle frontal gyrus orbit of a mouse of CPTX, and the brain section Alexa594 fluorescence imaging result for confirming the introduction position. It is a figure which shows the 3 chamber social interaction (social preference) test result. (A) is a diagram showing the results of a three-chamber social interaction (social recognition) test, and (b) is a diagram showing a test device. It is a figure which shows the prepulse inhibition test result. It is a figure which shows the forced swimming test result. It is a figure which shows the forced swimming test result.

Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention. ..

(AMPA receptor and its gene)
The AMPA receptor has four subunits, GluA1, GluA2, GluA3 and GluA4, which form homo or heterotetramers.
The coding regions (CDS) of mouse GluA1, mouse GluA2, mouse GluA3 and mouse GluA4 have the sequences represented by SEQ ID NOs: 1 to 4 in the sequence listing below.
The CDSs of human GluA1, human GluA2, human GluA3 and human GluA4 have the sequences represented by SEQ ID NOs: 5-8 below.

(Acquisition of AMPA receptor gene)
The method for obtaining the AMPA receptor gene is not particularly limited. Appropriate probes and primers were prepared based on the information on the base sequence and amino acid sequence of the AMPA receptor, and were prepared using them in a human cDNA library (appropriate cells expressing the AMPA receptor gene according to a conventional method). The AMPA receptor gene can be isolated by selecting the desired clone from the above.

<Method for producing a model non-human animal for mental illness or neurological disease caused by an increase or decrease in AMPA receptor gene expression in a predetermined region in the brain or an increase or decrease in AMPA receptor expression or function>
The method for producing a model non-human animal for a psychiatric disorder or a neurological disorder according to the first aspect (hereinafter, also simply referred to as “the production method according to the first aspect”) is a method.
A method for producing a model non-human animal of a mental disease or a neurological disease caused by an increase or decrease in AMPA receptor gene expression in a predetermined region in the brain or an increase (improvement) or decrease in AMPA receptor expression or function. ,
A step of reducing or increasing (improving) at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the above-mentioned predetermined region of a non-human animal and the function of the AMPA receptor as compared with the wild type. , Or a step of suppressing (blocking) or activating a neural pathway (nerve transmission pathway) connecting the predetermined region and another region (brain region other than the predetermined region).

The predetermined areas include the cerebellum, the frontal cortex (eg, anterior cingulate gyrus, middle frontal gyrus, etc.), and other cerebral cortex (eg, motor area, somatic sensory area, associative area, thalamus, auditory area, broker). Field, Wernicke field, cingulate gyrus, etc.), striatum, hippocampus, thalamus, thalamus, pineapple, pituitary gland, mesencephalon, posterior brain and cingulate gyrus. At least one selected from the group consisting of cerebellum, frontal lobe, parietal lobe, occipital lobe, and cingulate gyrus is preferred.

The above-mentioned psychiatric disorders or neurological disorders include (a) depression, major depression, bipolar depression, dysthymia, emotional disorders, recurrent depression, postpartum depression, winter depression (seasonal emotional disorder), etc. Stress disorder, depressive symptoms, manic disorder, anxiety, general anxiety disorder, anxiety syndrome, panic disorder, fear, social fear, social anxiety disorder, compulsive disorder, post-traumatic stress syndrome, post-traumatic stress disorder , Tauret Syndrome, Autism, Vulnerable X Syndrome, Let Syndrome, Adaptation Disorder, Bipolar Disorder, Neurology, schizophrenia, Chronic Fatigue Syndrome, Anxiety, Obstructive Disorder, Depressive Disorder, Depression, Psychiatric disorders such as divine hypersensitivity, attention deficit hyperactivity disorder, learning disorder, psychotic major depression, intractable major depression, treatment-resistant depression, drug addiction, alcohol dependence, gambling disorder, etc.
(B) Alzheimer's disease, Alzheimer's senile dementia, Parkinson's disease, Huntington's chorea, multiple cerebral infarct dementia, frontotemporal dementia, Parkinson's frontotemporal dementia, progressive nuclear paralysis, Pick syndrome, Niemann-Pick syndrome, cerebral cortical basal nucleus degeneration, Down's disease, defective dementia, Levy body dementia, muscle atrophic spinal lateral sclerosis, motor neurogenic disease, Kreuzfeld-Jakob disease, cerebral palsy, progression Neurodegenerative diseases such as cervical palsy, multiple sclerosis, dystonia, dementia, etc.
(C) Age-related cognitive and memory disorders such as age-related memory disorders and senile dementia.
(D) Intrinsic sleep disorders, extrinsic sleep disorders, circadian rhythm disorders, sleep contingencies, sleep disorders associated with internal or psychiatric disorders, stress insomnia, insomnia, insomnia neuropathy, sleep Sleep disorders such as insomnia,
(E) Respiratory depression caused by anesthetics, traumatic diseases, neurodegenerative diseases, etc. may be mentioned.
The above mental or neurological disorders are selected from the group consisting of schizophrenia, autism, epilepsy, depression, cerebral ischemia, Parkinson's disease, Alzheimer's disease, attention hyperactivity disorder (ADHD) and multiple sclerosis. At least one psychiatric or neurological illness is preferred, and more preferably at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism.

The above-mentioned increase is a statistically significant increase (for example, excess) with respect to the expression level or activity value when the subject was previously healthy, or a known range known as a normal value of the expression level or activity value. However, there is no particular limitation, but 1.2 with respect to the expression level or activity value when the subject was healthy, or the known range known as the normal value of the expression level or activity value. It is preferably fold or more, 1.4 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more, and particularly preferably 3 times or more. It is preferably 5 times or more, and most preferably 5 times or more.
The upper limit of the increase is not particularly limited, but may be 20 times or less, 10 times or less, 8 times or less, and the like.

The above-mentioned decrease refers to the expression level or activity value when the subject (for example, patient, non-human animal, etc.) was previously healthy, or the known range known as the normal value of the expression level or activity value. There is no particular limitation as long as it is a statistically significant decrease (for example, excessive decrease), but it is known as the expression level or activity value when the subject was healthy before, or the normal value of the expression level or activity value. It is preferably 3/4 or less, more preferably 1/2 or less, further preferably 1/4 or less, and particularly preferably 1/10 or less, based on the known range. Most preferably, there is no expression or activity.

A step of reducing or increasing at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the above-mentioned predetermined region of a non-human animal and the function of the AMPA receptor as compared with the wild type, or the above-mentioned step. The non-human animal subjected to the step of suppressing or activating the neural pathway connecting the predetermined region and the other region is not particularly limited as long as it is a non-human animal generally used as an experimental animal. It is preferably a non-human vertebrate, more preferably a non-human mammal, and even more preferably a rodent. Specific examples thereof include mice, rats, guinea pigs, hamsters, rabbits, cows, sheep, goats, pigs, horses, dogs, chickens, monkeys, etc., preferably mice, rats, guinea pigs, or hamsters, and more preferably. Is a mouse or rat, particularly preferably a mouse.
In that case, the strain of the mouse may be C57BL / 6 or BALB / c strain, but it is preferable that the gene background is closer to the pure strain by backcrossing or the like, and it is preferable that the strain is the same as that of the wild-type mouse to be compared.

At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region of the non-human animal and the function of the AMPA receptor in the above-mentioned step of decreasing or increasing the amount as compared with the wild type. As a method for reducing or increasing the amount as compared with the wild type, specifically, at least one substance selected from the group consisting of the following (1) to (4) is placed in the above-mentioned predetermined region in the brain stereotactically in the brain. Examples thereof include a method of internal injection (preferably intraventricular injection).
(1) AMPA receptor gene or a substance that reduces the expression of AMPA receptor,
(2) Substances that reduce the function of AMPA receptors,
(3) A substance that increases the expression of the AMPA receptor gene or AMPA receptor and (4) a substance that increases the function of the AMPA receptor The dose varies depending on the weight and age of the animal, the administration method, etc. A trader can appropriately select an appropriate dose.

Further, as a method of suppressing (blocking) or activating the neural pathway connecting the predetermined region and another region (brain region other than the predetermined region), active (excitable) or inhibitory DREADD (excitatory) or inhibitory DREADD ( The above-mentioned neural pathway is introduced into the above-mentioned neural pathway, for example, the above-mentioned neural pathway is introduced into the above-mentioned neural pathway.
For example, when the predetermined region is the anterior cingulate cortex (ACC), the other regions include other brain regions having a neural connection with the ACC, and more specifically, the claustrum, thalamus, and insular cortex. And so on.
When the predetermined region is the medial prefrontal cortex, examples of the other region include other brain regions having a neural connection with the medial prefrontal cortex, and more specifically, the amygdala basolateral nucleus and the like. ..
Examples of the active or inhibitory DREADD or RASSL include hM4Gi, hM3Gq, hM4Di, mAChR and the like.
Examples of the method of introduction include introduction using a viral vector (for example, an adeno-associated virus vector), and examples thereof include a method of administering the viral vector to the other region (for example, stereotactic injection administration in the brain). ..
The introduction amount varies depending on the body weight and age of the animal, the administration method, and the like, but those skilled in the art can appropriately select an appropriate introduction amount.

The active or inhibitory DREADD or RASSL can be activated by any agonist, eg, by administration of any agonist to the other regions mentioned above (eg, stereotactic injection in the brain). be able to.
Examples of the agonist include clozapine-N-oxide (CNO) and the like.
The dose varies depending on the body weight and age of the animal, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.

The above-mentioned suppression is known as the expression level or activity value related to neural connection when the subject (for example, patient, non-human animal, etc.) was healthy, or the normal value of the expression level or activity value. There is no particular limitation as long as it is statistically significant suppression (for example, excessive suppression) with respect to the range, but the expression level or activity value, or the expression level or activity value when the subject was previously healthy. It is preferably 3/4 or less, more preferably 1/2 or less, further preferably 1/4 or less, and 1/10 or less of the known range known as a normal value. Is particularly preferable.

The above activation is statistically significant with respect to the expression level or activity value related to neural connection when the subject was previously healthy, or the known range known as the normal value of the expression level or activity value. There is no particular limitation as long as it is activated (for example, excessive activation), but it is known as an expression level or activity value when the subject was previously healthy, or a normal value of expression level or activity value. With respect to the range, it is preferably 1.2 times or more, more preferably 1.4 times or more, further preferably 1.5 times or more, particularly preferably 2 times or more, 3 It is particularly preferable that the amount is double or more, and most preferably 5 times or more.
The upper limit of the activation is not particularly limited, and examples thereof include 20 times or less, 10 times or less, 8 times or less, and the like.

It is preferable that the above-mentioned step of reducing the amount as compared with the wild-type is a step of knocking down the above-mentioned AMPA receptor gene.
Here, knockdown of the AMPA receptor gene means an operation of reducing the transcription or expression of the AMPA receptor gene, and knockout of the AMPA receptor gene means disruption of the AMPA receptor gene.

Examples of the above-mentioned (1) AMPA receptor gene or substance that reduces the expression of AMPA receptor include nucleic acids having RNAi action (for example, siRNA, shRNA), antisense oligonucleotides, artificial nucleases, and the like, which are described below.
Regarding (2) a substance that lowers the function of the AMPA receptor, (3) a substance that increases the expression of the AMPA receptor gene or the AMPA receptor, and (4) a substance that increases the function of the AMPA receptor. It will be described later.

(Nucleic acid with RNAi action)
Examples of the above-mentioned (1) AMPA receptor gene or a substance that reduces the expression of the AMPA receptor include a nucleic acid having an RNAi action capable of suppressing the expression of the AMPA receptor gene. The nucleic acid having RNAi action preferably contains a continuous partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene or a sequence complementary thereto.
RNAi (RNA interference) refers to a phenomenon in which the expression of a target gene is suppressed when RNA (double stranded RNA: dsRNA) in which a part of mRNA encoding a part of a certain target gene is double-stranded is introduced into a cell. ..
Examples of the nucleic acid having an RNAi action include siRNA (small interfering RNA), shRNA (small hairpin RNA) and the like. Hereinafter, siRNA and shRNA will be described.

(A) siRNA
Examples of the siRNA include double-stranded RNA capable of suppressing the expression of the AMPA receptor gene by the action of RNAi or DNA encoding the above-mentioned double-stranded RNA, and suppressing the expression of the AMPA receptor gene by the action of RNAi. A double-stranded RNA having a continuous partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene or a DNA encoding the double-stranded RNA is preferable.
More specifically, the siRNA may have a sequence complementary to the number of consecutive sequences required to suppress the expression of the AMPA receptor gene in the base sequence of RNA transcribed from the AMPA receptor gene. Typically, double-stranded RNA containing at least 17 consecutive nucleotides in the base sequence of RNA transcribed from the AMPA receptor gene or DNA encoding the double-stranded RNA is preferred and transcribed from the AMPA receptor gene. It is more preferably a double-stranded RNA containing at least 19 consecutive nucleotides in the base sequence of the RNA to be obtained, or a DNA encoding the above double-stranded RNA, and more preferably in the base sequence of the RNA transcribed from the AMPA receptor gene. It is more preferably a double-stranded RNA containing at least 21 contiguous nucleotides or a DNA encoding the double-stranded RNA.
The siRNA is preferably a double-stranded RNA containing 30 or less consecutive nucleotides in the base sequence of RNA transcribed from the AMPA receptor gene, or a DNA encoding the double-stranded RNA.

Examples of the DNA encoding the double-stranded RNA include DNA having an inverted repeat sequence of a partial sequence of the AMPA receptor gene.
By introducing a DNA having such an inverted repeat sequence into a mammalian cell, the inverted repeat sequence of a partial sequence of the AMPA receptor gene can be expressed in the cell, thereby receiving AMPA by RNAi action. The expression of body genes can be suppressed.
An inverted repeat sequence is a sequence in which a partial sequence of an AMPA receptor gene and a complementary reverse sequence thereof are arranged in parallel via an appropriate sequence. Specifically, when the partial sequence of the AMPA receptor gene has a double strand consisting of the n base sequences shown below,
5'-X ₁ X ₂ . .. .. .. .. .. X _n-1 X _n -3'
3'-Y ₁ Y ₂ . .. .. .. .. .. Y _n-1 Y _n -5'
The inverted repeat has the following sequence.
5'-Y _n Y _n-1 . .. .. .. .. .. Y ₂ Y _1-3 '
3'-X _n X _n-1 . .. .. .. .. .. X ₂ X _1-5 '
(Here, among the bases represented by X and the bases represented by Y, those having the same subscript number are complementary bases to each other).

The inverted repeat sequence is a sequence in which the above two types of sequences are mediated by an appropriate sequence. There are two cases of inverted repeats, one is when the partial sequence of the target gene is upstream of the complementary sequence of the target gene, and the other is when the inverted sequence is upstream of the partial sequence of the complementary target gene. Can be considered. The inverted repeat sequence used in the present invention may be any of the above, but the inverted repeat is preferably located upstream of the partial sequence of the target gene complementary thereto.

(B) shRNA
As the shRNA, an inverted repeat sequence in which a partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene and an inverted sequence complementary thereto are paralleled via a sequence capable of forming a hairpin loop. Examples thereof include single-stranded RNA having or DNA encoding the above RNA.
The shRNA is suitable for a method of introducing a vector or virus expressing shRNA into a cell, and can function in the cell in the same manner as the siRNA described above.

For shRNA, the number required to suppress the expression of the AMPA receptor gene in the base sequence of the RNA transcribed from the AMPA receptor gene as a partial sequence in the base sequence of the RNA transcribed from the AMPA receptor gene. The sequence may be complementary to the contiguous sequence of, and typically a partial sequence containing at least 17 consecutive nucleotides in the base sequence of the RNA transcribed from the AMPA receptor gene is preferred. It is more preferably a partial sequence containing at least 19 consecutive nucleotides in the base sequence of the RNA to be produced, and it is a partial sequence containing at least 21 consecutive nucleotides in the base sequence of the RNA transcribed from the AMPA receptor gene. Is more preferable.
The partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene is preferably a partial sequence containing 30 consecutive nucleotides or less in the base sequence of RNA transcribed from the AMPA receptor gene.

The length of the sequence capable of forming the hairpin loop is not particularly limited as long as the hairpin loop can be formed, but is preferably 0 to 300 bp, more preferably 1 to 100 bp, still more preferably 2 to 75 bp, and particularly preferably 3 to 50 bp. Is. Restriction enzyme sites may be present in this sequence.

By incorporating the inverted repeat sequence of the target gene downstream of the promoter sequence that can be actuated in the mammal, the inverted repeat sequence of the target gene can be expressed in the intracellular cells of the mammal. The promoter sequence is not particularly limited as long as it can be operated in mammals.

In addition, the nucleic acid having RNAi action described above may be functionally bound to an appropriate terminator such as TPI1 terminator or ADH3 terminator for a human growth hormone terminator or a fungal host, if necessary. The recombinant vector also has elements such as polyadenylation signals (eg, from the SV40 or adenovirus 5E1b region), transcription enhancer sequences (eg, SV40 enhancers) and translation enhancer sequences (eg, encoding adenovirus VARNA). You may be doing it.
The recombinant vector or virus may further comprise a DNA sequence that allows the vector or virus to replicate within the packaging cell, an example of which is the SV40 origin of replication.
The recombinant vector or virus may further contain a selectable marker. Selectable markers include genes lacking its complement in packaging cells, such as, for example, dihydrofolate reductase (DHFR) or the sidosaccharomyces ponbe TPI gene, or, for example, ampicillin, kanamycin, tetracycline, chloramphenicol. , Neomycin or drug resistance genes such as dihydromycin.

Examples of the packaging cell for preparing the vector or virus by introducing the nucleic acid having the RNAi action described above or the vector or virus containing the nucleic acid thereof include higher eukaryotic cells, bacteria, yeasts, fungi and the like, but in mammalian cells. It is preferable to have.
A method for transforming a mammalian cell to express the gene introduced into the cell is also known, and for example, a lipofection method, an electroporation method, a calcium phosphate method and the like can be used.

For example, the nucleic acid having RNAi action described above can be administered alone or by injection or the like together with a transfection agent or a carrier for lipofection used to assist uptake into cells.
Examples of the carrier for lipofection include carriers having a high affinity for cell membranes (for example, liposomes, cholesterol, etc.), and lipofectamine or lipofectin is preferable, and lipofectamine is more preferable.

(Antisense oligonucleotide)
Examples of the above-mentioned (1) AMPA receptor gene or a substance that reduces the expression of the AMPA receptor include antisense oligonucleotides having a sequence complementary to a continuous sequence in the AMPA receptor gene.
For example, two hybrids produced by hybridizing an oligonucleotide contained in an AMPA receptor gene and the antisense oligonucleotide complementary thereto into cells (preferably in the nucleus) and then forming a hybrid. A strand-specific nuclease (eg, RNase H) can degrade the mRNA of an AMPA receptor containing a nucleotide chain and suppress transcription, translation, etc. of the AMPA receptor gene.
The antisense oligonucleotide may be DNA or RNA, but is preferably DNA from the viewpoint that mRNA is cleaved by the specific nuclease.
The antisense oligonucleotide may have a sequence complementary to the number of consecutive sequences required for suppressing the expression of the AMPA receptor gene in the base sequence of the AMPA receptor gene, and is typically used. Is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 10 consecutive nucleotides, preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 13 nucleotides. More preferred.
Further, as the upper limit of the base length of the antisense oligonucleotide, it is more preferable that the antisense oligonucleotide has a sequence complementary to a continuous oligonucleotide of 20 nucleotides or less in the base sequence of the AMPA receptor gene. , It is more preferably an antisense oligonucleotide having a sequence complementary to a contiguous 17-nucleotide or less oligonucleotide.

The antisense oligonucleotide is preferably an antisense oligonucleotide containing at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure.
In the antisense oligonucleotide, it is preferable that at least one of the nucleotides at one end (preferably 1 to 3 bases from the end) has a crosslinked structure or an alkoxy structure.
The antisense oligonucleotide can be produced by a conventional method using a DNA synthesizer and a known organic synthesis technique.

The intracellular (preferably intranuclear) uptake of the antisense oligonucleotide may be free uptake.
From the viewpoint of improving intracellular uptake, a carrier for lipofection may or may not be used.

In addition, as one embodiment of intracellular uptake of antisense oligonucleotide, the vector or virus is prepared after being inserted into a suitable vector or virus and further introduced into a suitable packaging cell to prepare the vector or virus. It may be an embodiment in which the virus infects the target cancer cells.
The type of the above-mentioned suitable vector or virus is not particularly limited, and may be, for example, an autonomously replicating vector or virus, but a chromosome integrated into the genome of the packaging cell when introduced into the packaging cell. It is preferable that it is duplicated with.

Examples of the packaging cell for preparing the vector or virus by introducing the antisense oligonucleotide or the vector or virus containing the same include higher eukaryotic cells, bacteria, yeasts, fungi and the like, but the cells are mammalian cells. Is preferable.

(Artificial nuclease)
Examples of the above-mentioned (1) AMPA receptor gene or a substance that reduces the expression of the AMPA receptor include artificial nucleases for genome editing such as CRISPR (Crust Generally Interspaced Short Palindromic Repeats) / Cas nuclease, and TranscrictionActivator-Li It may be an artificial restriction enzyme (artificial nuclease) using Nuclease (TALEN) and a zinc finger nuclease (ZFN).

The CRISPR / Casnuclease contains a guide RNA and a Casnuclease (preferably Cas9).
At least a partial sequence in the target AMPA receptor gene includes oligonucleotides contained in the AMPA receptor gene.

Transpose a composition containing a guide RNA or a DNA encoding a guide RNA specific to the AMPA receptor gene and a nucleic acid or Cas nuclease encoding a Cas nuclease into eukaryotic cells or eukaryotes containing the AMPA receptor gene. By effecting, the expression of the AMPA receptor gene can be reduced.
Nucleic acids or Casnucleases encoding Casnucleases and DNAs encoding guide RNAs or guide RNAs can be obtained from a variety of methods known in the art such as microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticles. It can be transferred into cells by, but is not limited to, intracellular transfection, protein transduction domain-mediated transduction, virus-mediated gene delivery, and PEG-mediated transfection into protoplasts. Also, the nucleic acid encoding the Cas nuclease or the Cas nuclease and guide RNA can be transferred into the organism by various methods known in the art for administering genes or proteins such as infusions. The nucleic acid or Cas protein encoding the Cas nuclease can be transferred into the cell in the form of a complex with a guide RNA or separately. Cas nuclease fused to a protein transduction domain such as Tat can also be efficiently delivered intracellularly.
Preferably, eukaryotic cells or eukaryotes are co-transfected or serially transfected with Cas9 nuclease and guide RNA.
Serial transfection can be performed first by transfection with a nucleic acid encoding Casnuclease, followed by a second transfection with a naked guide RNA. Preferably, the second transfection is after 3, 6, 12, 18, 24 hours, but is not limited to them.

((2) Substances that reduce the function of AMPA receptors, (3) Substances that increase the expression of AMPA receptor genes or AMPA receptors, and (4) Substances that increase the function of AMPA receptors)
The above (1) AMPA receptor gene or a substance that reduces the expression of the AMPA receptor is as described above.
Examples of the substance (2) that lowers the function of the AMPA receptor include an AMPA receptor antagonist, a negative allosteric modulator of the AMPA receptor, a nucleic acid such as an aptamer, and an antibody.
As the negative allosteric modulator of the AMPA receptor, any compound known as the negative allosteric modulator of the AMPA receptor can be used.
Examples of the AMPA receptor antagonist include CNQX, DNQX, NBQX, perampanel and the like.
An aptamer is a nucleic acid that is composed of single-stranded RNA or DNA and binds to a target protein by its three-dimensional structure to inhibit its function.
Aptamers have high binding and specificity to target proteins, low immunogenicity, can be produced by chemical synthesis, and have high storage stability.
Aptamers that selectively bind to AMPA receptors can be obtained by the SELEX (Systematic Evolution of Ligands by EXPonential evolution) method.
The antibody that selectively binds to the AMPA receptor may be either a polyclonal antibody or a monoclonal antibody as long as it can selectively bind to the AMPA receptor.

Examples of the above-mentioned (3) AMPA receptor gene or a substance that increases the expression of the AMPA receptor include a nucleic acid that knocks in the AMPA receptor gene, an artificial nuclease, and the like.
Examples of the substance (4) that increases the function of the AMPA receptor include an AMPA receptor agonist, a positive allosteric modulator of the AMPA receptor, a nucleic acid such as an aptamer, a synapse organizer, and an antibody.
Examples of the AMPA receptor agonist include glutamic acid, bromo-willardine, ibotenic acid, ACPA, kainic acid, domoic acid and the like.
Positive allosteric modulators of AMPA receptors include CX-516, CX-614, aniracetam, CTZ, IDR-21, diazoxide and the like.
Synapse organizers, which may be natural or artificial (synthetic), include chimeric molecules that link the Neurexin binding domain of Celeberin 1 (Cbln1) with the AMPA receptor binding domain of Neuronal Pentraxin 1. Cerebello-Pentraxin (CPTX) is preferred.

In the above step of decreasing or increasing the amount as compared with the wild type, the degree of the decrease is not particularly limited as long as it is a statistically significant decrease, but the AMPA receptor gene or AMPA is compared with the wild type non-human animal. The expression of the receptor or the function of the AMPA receptor is preferably 3/4 or less, more preferably 1/2 or less, further preferably 1/4 or less, and 1/10 or less. Is particularly preferable, and it is most preferable that the expression or function is lost.

The degree of the increase is not particularly limited as long as it is a statistically significant increase, but the expression of the AMPA receptor gene or the AMPA receptor or the function of the AMPA receptor is higher than that of the wild type non-human animal. It is preferably 1.2 times or more, more preferably 1.4 times or more, further preferably 1.5 times or more, particularly preferably 2 times or more, and 3 times or more. It is particularly preferable, and it is most preferable that it is 5 times or more.
The upper limit of the increase is not particularly limited, but may be 20 times or less, 10 times or less, 8 times or less, and the like.

Based on the base sequence information of the AMPA receptor gene, the expression of the AMPA receptor gene in various animal tissues can be detected even in Incilico. In vivo and in vitro, for example, by using a probe or primer having a part or all of the base sequence of the gene, the expression of the AMPA receptor gene in various animal tissues can be detected. The expression of the AMPA receptor gene can be detected by a conventional method such as RT-PCR, Northern blot, Southern blot and the like.
Further, the expression level of the AMPA receptor gene at the mRNA level can also be measured by a conventional method such as RT-PCR, Northern blot, Southern blot or the like.

When performing PCR, the primer is not particularly limited as long as it can specifically amplify only the AMPA receptor gene, and can be appropriately set based on the sequence information of the AMPA receptor gene. For example, an oligonucleotide containing at least 10 consecutive nucleotides in the base sequence of the AMPA receptor gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as a probe or primer. More specifically, an oligonucleotide having a continuous 10 to 60 residue, preferably 10 to 40 residue base sequence in the base sequence of the AMPA receptor gene, and an anti having a sequence complementary to the oligonucleotide. Sense oligonucleotides can be used.

The above-mentioned oligonucleotide and antisense oligonucleotide can be produced by a conventional method using a DNA synthesizer. As the oligonucleotide or antisense oligonucleotide, for example, in a part of the base sequence of the mRNA to be detected, a sense primer corresponding to the base sequence on the 5'end side, an antisense primer corresponding to the base sequence on the 3'end side, and the like. Can be mentioned. The sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and number of bases do not change drastically, and examples thereof include those having about 10 to 60 bases and those having about 10 to 40 bases. Is preferable. Further, in the present invention, the above-mentioned derivative of the oligonucleotide can be used, and for example, a methyl form or a phosphorothioate form of the oligonucleotide can be used.

The expression level of the AMPA receptor protein can be measured by Western blotting using an antibody described later or by ordinary immunoassay such as ELISA. Specifically, it can be carried out by a conventional method known to those skilled in the art described in Molecular Cloning 2nd Edition or Current Protocols in Molecular Biology and the like.

<Mental illness model non-human animal>
A model non-human animal for a mental or neurological disorder according to the second aspect (hereinafter, also simply referred to as “model animal according to the second aspect”) has increased expression of the AMPA receptor gene in a predetermined region in the brain. Or a non-human animal model of a psychiatric or neurological disorder resulting from a decrease or an increase or decrease in the expression or function of the AMPA receptor.
At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor is decreased or increased (improved) as compared with the wild type, or the above. The neural pathways that connect a predetermined region to another region are suppressed or activated.
Examples of the predetermined region include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
Examples of the above-mentioned psychiatric disorder or neurological disorder include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
Examples of the non-human animal used for producing the model animal according to the second aspect include the same as the above-mentioned specific examples and preferable examples for the production method according to the first aspect.

The first aspect is as a method for reducing or increasing at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor as compared with the wild type. As described above, at least one substance selected from the group consisting of the above (1) to (4) is stereotactically injected into the predetermined region of the brain (preferably the brain). The method of indoor injection) and the like can be mentioned.
(1) A substance that reduces the expression of the AMPA receptor gene or AMPA receptor, (2) a substance that reduces the function of the AMPA receptor, and (3) a substance that increases the expression of the AMPA receptor gene or the AMPA receptor. Specific examples and preferable examples of the substance and the substance that increases the function of the above-mentioned (4) AMPA receptor include the same as the above-mentioned specific example and preferable example for the production method according to the first aspect.
As a method for suppressing or activating a neural pathway connecting the predetermined region and another region, as described above for the production method according to the first aspect, an active or inhibitory DREADD or RASSL is used. Examples thereof include a method of introducing into the above-mentioned other region where a nerve pathway exists, and specific examples and preferable examples include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.

The degree of the decrease is not particularly limited as long as it is a statistically significant decrease, and examples of the production method according to the first aspect are the same as those of the above-mentioned specific examples and preferable examples.
The degree of the increase is not particularly limited as long as it is a statistically significant improvement, and examples of the production method according to the first aspect are the same as those of the above-mentioned specific examples and preferable examples.

As the model animal according to the second aspect, at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor and the function of the AMPA receptor in the frontal lobe, the occipital lobe, the cerebellum or the zonal gyrus is selected. It is reduced or reduced compared to the wild type of the non-human animal.
At least one selected from the group consisting of AMPA receptor gene or AMPA receptor expression and AMPA receptor function in the parietal lobe, occipital lobe, or middle frontal gyrus fossa is a wild-type non-human animal. It is preferably a model non-human animal that has been increased (improved) as compared to the model.
When the psychiatric or neurological disorder is at least one psychiatric disorder selected from the group consisting of bipolar disorder and schizophrenia, the AMPA receptor gene or AMPA receptor in the frontal lobe, occipital lobe, cerebellum or zonal gyrus. It is preferred that at least one selected from the group consisting of expression and function of the AMPA receptor is reduced as compared to the wild type.
When the above mental or neurological disorder is autism, select from the group consisting of the expression of AMPA receptor gene or AMPA receptor and the function of AMPA receptor in the parietal lobe, occipital lobe, or mid-frontal gyrus. It is preferable that at least one of these is increased (improved) as compared with the wild type.
It is preferred that the reduced animal is an animal in which the AMPA receptor gene has been knocked down.

<Method of screening for preventive or therapeutic agents for mental or neurological disorders>
In the screening method according to the third aspect, a test substance is administered to the non-human animal, and a preventive or therapeutic agent for the mental disease or the neurological disease is used as an index for the prevention, suppression or cure of the mental disease or the neurological disease in the non-human animal. Is a method of screening.
Examples of the above-mentioned psychiatric disorder or neurological disorder include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
In the screening method according to the third aspect, the screening means to at least narrow down the population of the test substance using the above as an index.
The screening method according to the third aspect is to determine whether or not the lesion occurs after the above-mentioned administration as compared with the absence of the test substance (for example, a system before administration of the test substance or a negative control system). Alternatively, it is preferable to include a screening step of selecting the preventive or therapeutic agent candidate by judging the action of the test substance by observation using whether or not the lesion is healed or relieved as an index.
After the above administration, it is possible to judge the action of the test substance by observing whether or not the lesion occurs, or whether or not the lesion is healed or relieved, for example, in the Posalt forced swimming test. It can be performed by evaluating the tail suspension test, the open field test, the rotarod test, the prepulse inhibition test, the 3-chamber social interaction test, the life span, the weight, and the like.

The test substance can be administered by a method known to those skilled in the art such as intracerebral injection (preferably intracerebroventricular injection), oral administration, intraarterial injection, intravenous injection, intraperitoneal injection, and subcutaneous injection. .. For example, the above-mentioned predetermined regions in the brain (for example, cerebellum, prefrontal cortex, other cerebral cortex, striatum, hippocampus, thorax, hypothalamus, pineapple, pituitary gland, middle brain, posterior brain, choroidal flora, etc.) Preferably, intracerebral injection (preferably intraventricular injection) is performed stereotactically into the cerebellum, frontal lobe, parietal lobe, occipital lobe, or band-shaped circumflex.
For example, when the non-human animal is a bipolar disorder model animal, it is preferable to inject it into the cerebellum in a stereotactic manner.
When the above-mentioned non-human animal is a schizophrenia model animal, it is preferable to perform intracerebral injection stereotactically in the anterior cingulate gyrus.
When the non-human animal is an autism model animal, it is preferable to inject it into the middle frontal gyrus orbit in a stereotactic manner.
When the non-human animal is a depression model animal, it is preferable to inject it into the medial prefrontal cortex stereotactically in the brain as the other region.

Any substance can be used as the test substance used in the screening method according to the third aspect. The type of the test substance is not particularly limited, and may be a nucleic acid molecule, an antibody, a synthetic peptide (oligo or polypeptide), an individual small molecule synthetic compound, or is present in a natural product extract. It may be a compound or the above-mentioned artificial nuclease for genome editing. Alternatively, the test compound may also be a synthetic peptide (oligo or polypeptide) library (eg, a random peptide library with 10 or less amino acid residues), a compound library, a phage display library or a combinatorial library. Construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.
The test substance is preferably a random peptide library with 10 or less amino acid residues, a low molecular weight compound (eg, a compound library), a nucleic acid molecule, an artificial nuclease or antibody for genome editing, and an AMPA receptor gene or AMPA receptor. From the viewpoint of high specificity for the nucleic acid molecule or antibody, a nucleic acid molecule or antibody is more preferable, and an aptamer or antibody that selectively binds to the nucleic acid molecule or AMPA receptor having a sequence complementary to the oligonucleotide contained in the AMPA receptor gene. Is more preferable.

Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples.

<Example 1> Manufacture of a bipolar disorder model mouse Based on the findings that the present inventors have found that the amount of AMPA receptors in the cerebellum decreases in a manic state based on the YMRS evaluation scale of human bipolar disorder patients. A bipolar disorder model mouse was manufactured as follows.
(Method)
FIG. 1 is a brain atlas showing the coordinates of introduction of shRNA into the cerebellum of mice for AMPA receptors.
Lentivirus expressing shRNA capable of selectively suppressing the expression of AMPA receptors (GluA1-4) or scrambled RNA (non-functional shRNA as a control) is shown in FIG. 1 using a glass micropipette. As shown in Table 1 below, 500 nl (0.5 μl) was injected stereotactically into the brain coordinates of the mouse cerebellum (6 sites / animal in total).

Here, the AP coordinates represent the anterior distance from the bregma suture in the brain map.
The ML coordinates represent the distances from the median to the left and right sides of the brain map.
The H coordinate represents the depth from the bregma suture in the brain map.

After collecting the brain by perfusion fixation (under anesthesia) using PFA solution (paraformaldehyde fixative), prepare frozen sections and confirm the fluorescence of GFP (the above lentivirus expresses GFP as well as shRNA). As a result of identifying the virus introduction site, it was confirmed that the virus was introduced over a wide area of the cerebellum by the above injection.

The obtained knockdown mice were subjected to a posalt forced swimming test, a tail suspension test, an open field test, and a rotarod test, and evaluated as bipolar disorder model mice.
The Posalt forced swimming test and the tail suspension test are known as despair models of depression, and the onset time of immobile behavior can be evaluated as depression-like behavior.

(Posalt forced swimming test)
A posalt forced swimming test was performed on the knockdown mouse (n = 16) and the mouse (n = 12) into which a scrambled sequence shRNA (non-functional shRNA) was introduced as a control. The results are shown in FIG.
As is clear from the results shown in FIG. 2 (a), the group of mice introduced with shRNA for the AMPA receptor (n = 16) was depressed as compared with the group of control scrambled sequence shRNA-introduced mice (n = 12). It can be said that the immobility time, which is a like behavior, is significantly reduced, that is, the manic state is significantly increased, which is suitable as an animal model of bipolar disorder.
As is clear from the results shown in FIG. 2 (b), it was confirmed that the number of immobilities, which is a change in behavior, is not affected by knockdown (decrease in AMPA receptors in the cerebellum).

(Tail suspension test)
A tail suspension test was performed on the above-mentioned knockdown mouse (n = 16) and a mouse (n = 12) into which a scrambled sequence shRNA (non-functional shRNA) was introduced as a control. The results are shown in FIG.
From the results shown in FIG. 3 (a), although it was not remarkable as compared with the forced swimming test, the mouse group (n = 12; 4 out of 16 mice) into which shRNA for the AMPA receptor was introduced went up to the bar. (Excluded) is a depressive behavior in the tail suspension test, reduced immobility time compared to the control scrambled sequence shRNA-introduced mouse group (n = 9; excluded because 3 out of 12 mice went up to the bar). It showed a tendency. That is, the manic state tends to increase, and it can be said that it is appropriate as an animal model of bipolar disorder.
As is clear from the results shown in FIG. 3 (b), it was confirmed that the number of immobilities, which is a change in behavior, was not affected by knockdown (decrease in AMPA receptors in the cerebellum).

(Open field test)
When an open field test was conducted with the amount of spontaneous activity and anxiety-like behavior as evaluation items, the knockdown mouse (n = 16) and the mouse (n = 12) into which a scrambled sequence shRNA (non-functional shRNA) was introduced as a control were introduced. ) Was not significantly different. This result means that the decrease in akinesia observed in the forced swimming test and the tail suspension test is not merely a side effect of increased activity.

(Rotarod test)
When a rotarod test was performed with motor learning and coordinated movement as evaluation items, the knockdown mouse (n = 16) and the mouse (n = 12) into which a scrambled sequence shRNA (non-functional shRNA) was introduced as a control were used. No significant difference was found between the two. It is known at the textbook level that the function of the cerebellum is related to "learning and control of movement", but the decrease in AMPA receptors in the cerebellum in this model causes motor dysfunction that can be detected by the rotarod test. It was shown not to cause.

From the above-mentioned Posalt forced swimming test, tail suspension test, open field test, and rotarod test results, it can be said that the obtained knockdown mouse is suitable as a bipolar disorder model mouse.

<Example 2> Production of schizophrenia model mice Based on the findings that the present inventors have found that AMPA receptor density is relatively decreased in the anterior cingulate gyrus in human schizophrenia patients, schizophrenia. A schizophrenia model mouse was produced as follows.
(Method)
FIG. 4 is a synthetic diagram of a brain map showing the coordinates of introduction of shRNA into the anterior cingulate gyrus of AMPA receptors and the results of GFP fluorescence imaging of brain sections for confirming the introduction position.
Lentivirus expressing shRNA capable of selectively suppressing the expression of AMPA receptors (GluA1-4) or scrambled RNA (non-functional shRNA as a control) is shown in FIG. 4 using a glass micropipette. As described above, 300 nl (0.3 μl) was introduced stereotactically into the brain coordinates of the following (1) and (2), which are the anterior cingulate cortex of the mouse.
(1) AP coordinates 1.2 mm, ML coordinates ± 0.5 mm, and H coordinates 1.6 mm.
(2) AP coordinates 0 mm, ML coordinates ± 0.5 mm, and H coordinates 1.2 mm.

The obtained knockdown mice were evaluated as schizophrenia model mice by performing a prepulse inhibition (hereinafter, also simply referred to as “PPI”) test, an open field test, and a three-chamber social interaction test.
Here, the prepulse inhibition refers to a phenomenon in which a startle response is significantly suppressed by a weak stimulus (prepulse) preceding immediately before the startle stimulus (pulse), and is used in schizophrenia patients and schizophrenia. It can be used as an endophenotype for schizophrenia because it shows a decline in both animal models.

(PPI test)
From 3 weeks to 6 days after the introduction of the wrench virus, acclimatization to the PPI test device and mouse handling were performed for 10 minutes each.
The PPI test was performed on the 7th day from the start of acclimatization to the above PPI test apparatus, and the Inhibition ratio was calculated.
The Inhibition ratio was calculated in "pleplese74dB pulse120dB", "plepulus78dB pulse120dB" and "plepulus84dB pulse120dB".
As is clear from the results shown in FIG. 5, in the "plepulus74dB pulse120dB" and "plepulus78dB pulse120dB", the mice introduced with the control scrambled RNA were compared with the mouse group (n = 10) in which the shRNA for the AMPA receptor was introduced. In the group (n = 10), suppression by prepulse occurred significantly, that is, it did not occur significantly in the mouse group in which shRNA for AMPA receptor was introduced, and it can be said that it is suitable as an animal model of schizophrenia. ..

(Open field test)
An open field test was performed 3 days after mouse handling and initiation of acclimatization to the PPI test device.
Behavioral activity in a 50 cm × 50 cm open field was followed for 60 minutes using imaging software. The results are shown in FIG.
As is clear from the results shown in FIG. 6, the rate of decrease in the travel distance after 30 minutes is significantly higher in the group of mice introduced with shRNA for the AMPA receptor than in the group of mice introduced with the control scrambled RNA. It can be said that the low adaptability to the novel environment is low, that is, the travel distance is significantly long even after 30 minutes, and the mouse group in which shRNA for AMPA receptor is introduced has low adaptability to the novel environment.

(3-chamber social interaction test)
The 3-chamber social interaction (social preference) test was performed one week after the above PPI test. Specifically, after acclimatization to the test device (see FIG. 7B) for 5 minutes,
The residence time of the test mice in the chamber without other individuals (empty chamber) and the chamber with other individuals (stranger chamber) was measured for 10 minutes. The results are shown in FIG. 7 (a).
As is clear from the results shown in FIG. 7 (a), in the mouse group into which the control scrambled RNA was introduced, the residence time in the stranger chamber was significantly longer than that in the empty chamber, and the rate of approaching other individuals was high. It can be said that the sex is high.
On the other hand, in the mouse group into which shRNA for AMPA receptor was introduced, there was no significant difference between the staying time in the empty chamber and the staying time in the stranger chamber, the rate of approaching other individuals decreased, and the sociality was low. I can say.

After the 3-chamber social interaction test was completed, the brain was collected by perfusion fixation (under anesthesia) using PFA solution. Then, a frozen section was prepared and the fluorescence of GFP was confirmed (the above-mentioned lentivirus expresses GFP together with shRNA) to identify the virus introduction site. The results are shown in the composite diagram of FIG.

From the above PPI test, open field test and 3-chamber social interaction test results, it can be said that the obtained knockdown mouse is suitable as a schizophrenia model mouse.

<Example 3> Production of autism model mouse Based on the findings that the present inventors have found that AMPA receptor density increases in the middle frontal gyrus orbit in human autism patients, autism. The model mouse was manufactured as follows.
(Method)
FIG. 8 is a composite diagram of a brain map showing the coordinates of introduction of CPTX into the middle frontal gyrus orbital part of a mouse and a brain section Alexa594 fluorescence imaging result for confirming the introduction position.
To increase AMPA receptors, CPTX, a synthetic synapse organizer, or vehicle (solvent) as a control, using a glass micropipette, is the middle frontal gyrus orbitofrontal cortex of mice as shown in FIG. 8 (3). ), 300 ln (0.3 μl) was introduced per location in a stereotactic manner.
(3) AP coordinates 2.5 mm, ML coordinates ± 1.3 mm, and H coordinates 1.9 mm.

Mice to which CPTX was administered to the middle frontal gyrus orbit were evaluated as autism model mice by conducting a 3-chamber social interaction test and a PPI test.

(3-chamber social interaction (social preference) test 1)
The 3-chamber social interaction (social preference) test 1 was carried out one week after the introduction. Specifically, after acclimatization to the test equipment for 5 minutes,
The residence time of the test mice in the chamber without other individuals (empty chamber) and the chamber with other individuals (stranger1 chamber) was measured for 10 minutes. The results are shown in FIG.
As is clear from the results shown in FIG. 9, in the mouse group (n = 12) into which the control vehicle was introduced, the residence time in the stranger1 chamber was significantly longer than that in the empty chamber, and the rate of approaching other individuals was high. It can be said that it is highly social.
On the other hand, in the mouse group (n = 12) in which CPTX was introduced into the middle frontal gyrus orbit, there was no significant difference between the staying time in the empty chamber and the staying time in the stranger1 chamber, and the rate of approaching other individuals decreased. However, it can be said that the sociality is low.

(3-chamber social interaction (social cognition) test 2)
After that, another individual (stranger2) is placed in the above chamber, and the staying time between the chamber containing the new individual stranger2 and the contacted stranger1 chamber is measured for 10 minutes, and the three-chamber social interaction (social recognition) is performed. ) Test 2 was performed. The results are shown in FIG.
As is clear from the results shown in FIG. 10, in the mouse group introduced with the control vehicle, the residence time in the chamber containing the new individual stranger 2 is significantly longer than that in the chamber of the contacted stranger 1, and the new individual approaches the new individual. It can be said that the ratio is high and social recognition is high.
On the other hand, in the mouse group in which CPTX was introduced into the middle frontal gyrus orbit, there was no significant difference between the staying time in the chamber of the new individual's stranger2 and the staying time in the chamber of the contacted stranger1, and the new individual was approached. It can be said that the proportion of mice is decreasing and social awareness is low.

(PPI test)
When the PPI test was performed in the same manner as in Example 2, there was no significant difference between the control vehicle-administered group and the CPTX-administered group in any of "plepulus 74 dB pulse 120 dB", "plepulus 78 dB pulse 120 dB" and "plepulus 84 dB pulse 120 dB".

After the PPI test was completed, the brain was collected by perfusion fixation (under anesthesia) using PFA solution. Then, frozen sections were prepared, and the site of introduction of CPTX was identified by using an immunohistochemical method using an antibody against His-tag (the above-mentioned CPTX was added with His-tag). The results are shown in the composite diagram of FIG.

From the results of the three-chamber social interaction test and the PPI test, it can be said that the mouse to which CPTX was administered to the middle frontal gyrus orbit is suitable as an autism model mouse.

<Example 4> Production of schizophrenia model mouse A schizophrenia model mouse was produced as follows.
HM4Gi, which suppresses neural function using adeno-associated virus, was introduced bilaterally into the anterior cingulate cortex (ACC), which is the causative region, and claustrum, which is the brain region that has neural connections.
Three weeks after the introduction of the adeno-associated virus, hM4Gi was activated with clozapine-N-oxide (CNO) after acclimatization to the PPI test apparatus, and the PPI test was carried out in the same manner as in Example 2 to calculate the Inhibition ratio.
Then, on 5 to 7 days, physiological saline was administered to the same individual, and the same test was performed. The results are shown in FIG.

As is clear from the results shown in FIG. 11, in all of "plepulus74dB plus120dB", "plepulus78dB plus120dB" and "plepulus84dB plus120dB", nerve connection was suppressed in claustrum as compared with the control group to which physiological saline was administered. In the group of mice with schizophrenia, the Inhibition ratio was significantly reduced, and it can be said that it is suitable as an animal model of schizophrenia.

<Example 5> Production of depression model rat A depression model rat was produced as follows.
As the rat, a 6-week-old male Wistar rat (manufactured by Charles River) was used. Short hairpin (sh) RNA that specifically suppresses the expression of AMPA receptor subunits 1-3 (GluA1-3) using lentivirus in the bilateral medial frontal cortex of Wistar rats or GluA1-3 as a control. A scrambled RNA that does not suppress expression (non-functional shRNA as a control) was introduced.
(Forced swimming test)
One week after the introduction, a forced swimming test was conducted. The forced swimming test was carried out by injecting tap water having a water temperature of 26 ± 0.5 ° C. to a water level of 40 cm using a cylindrical cage having a diameter of 20 cm and a height of 50 cm. Rats were placed in this cylindrical cage and the state of swimming for 10 minutes was photographed using a digital video camera (HC-V750, manufactured by Panasonic). After shooting, the rat was wiped with a paper towel and returned to the home cage. In the analysis, in the acute administration experiment and the chronic administration experiment, the time during which the rat's limbs were not moved in 7 to 10 minutes of the captured video was measured as the immobility time. The results are shown in FIG.

As is clear from the results shown in FIG. 12, the rat (n = 6) introduced with shRNA that specifically suppresses the expression of the AMPA receptor subunit GluA1-3 is the rat (n = 6) introduced with the scrambled RNA. It can be said that it is suitable as an animal model of depression because it showed a significantly longer non-working time as compared with = 5).

<Example 6> Production of depression model rat A depression model rat was produced as follows.
As the rat, a 4-week-old male Wistar rat (manufactured by Charles River) was used. HM4Gi, which suppresses neural function using adeno-associated virus, was introduced bilaterally into the basolateral amygdala nucleus, which is a brain region with neural connections to the medial prefrontal cortex, which is the causative region.
Three weeks after the introduction of the adeno-associated virus, hM4Gi was activated by clozapine-N-oxide (CNO), a forced swimming test was performed in the same manner as in Example 5, and the immobility time was measured. The results are shown in FIG.

As is clear from the results shown in FIG. 13, the CNO-administered rat (n = 11) showed a significantly longer non-working time than the control saline-administered rat (n = 11), and thus depression. It can be said that it is suitable as an animal model of.

Claims (12)

1. A method for producing a model non-human animal for a psychiatric disorder or a neurological disorder caused by an increase or decrease in AMPA receptor gene expression in a predetermined region in the brain or an increase or decrease in AMPA receptor expression or function.
   A step of reducing or increasing at least one selected from the group consisting of the expression of an AMPA receptor gene or AMPA receptor in the predetermined region of a non-human animal and the function of the AMPA receptor as compared with the wild type, or the above-mentioned step. A method comprising the step of suppressing or activating a neural pathway connecting a predetermined region with another region.
2. At least the predetermined region is selected from the group consisting of cerebellum, prefrontal cortex, other cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, pineapple, pituitary gland, midbrain, posterior brain and choroidal flora. The method according to claim 1, which is one.
3. The method according to claim 2, wherein the predetermined region is at least one selected from the group consisting of the cerebellum, frontal lobe, parietal lobe, occipital lobe, and cingulate gyrus.
4. The mental or neurological disorders are bipolar disorder, schizophrenia, autism, epilepsy, depression, cerebral ischemia, Parkinson's disease, Alzheimer's disease, attention hyperactivity disorder (ADHD) and polysclerosis, sleep disorder. , And the method according to any one of claims 1 to 3, which is at least one psychiatric disorder or neurological disorder selected from the group consisting of circadian rhythm disorders.
5. The method according to claim 4, wherein the psychiatric disorder or neurological disorder is at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism.
6. The method according to any one of claims 1 to 5, wherein the step of reducing the amount as compared with the wild type is a step of knocking down the AMPA receptor gene.
7. A model non-human animal for psychiatric or neurological disorders resulting from increased or decreased expression of the AMPA receptor gene in a predetermined region of the brain or increased or decreased expression or function of the AMPA receptor.
   At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor is reduced or increased as compared with the wild type, or the predetermined region and the said region. A model non-human animal in which the neural pathways connecting to other regions are suppressed or activated.
8. The animal according to claim 7, wherein the predetermined region is at least one selected from the group consisting of the cerebellum, frontal lobe, parietal lobe, occipital lobe, and cingulate gyrus.
9. In the frontal lobe, occipital lobe, cerebral or cingulate gyrus, at least one selected from the group consisting of AMPA receptor gene or AMPA receptor expression and AMPA receptor function was reduced or reduced compared to the wild type. ,
   In the parietal lobe, occipital lobe, or middle frontal gyrus, at least one selected from the group consisting of AMPA receptor gene or AMPA receptor expression and AMPA receptor function is increased compared to the wild type. Or
   The animal according to claim 8, wherein the neural pathway connecting the anterior cingulate cortex and the claustrum is suppressed.
10. The animal according to any one of claims 7 to 9, wherein the psychiatric disorder or neurological disorder is at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism. ..
11. The animal according to any one of claims 7 to 10, wherein the lowered animal is an animal in which the AMPA receptor gene has been knocked down.
12. The test substance is administered to the animal according to any one of claims 7 to 11, and a prophylactic or therapeutic agent for the psychiatric disorder or the neurological disorder is screened using the prevention, suppression or cure of the psychiatric disorder or the neurological disorder in the animal as an index. how to.

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