WO2009093772A1

WO2009093772A1 - Plc beta4 mutant mice as a model for testing anxiety disorder drugs

Info

Publication number: WO2009093772A1
Application number: PCT/KR2008/001275
Authority: WO
Inventors: Hee Sup Shin; Sukchan Lee; Hui-Su Kim; Sukwoo Choi; Ingie Hong
Original assignee: Korea Institute Of Science And Technology
Priority date: 2008-01-23
Filing date: 2008-03-06
Publication date: 2009-07-30
Also published as: KR20090081232A; KR100979438B1

Abstract

The present invention relates to a use of a PLCβ4 (Phospholipaseβ4 ) knock-out animal, more precisely, a use or a method of using a PLCβ4 knock-out animal as an anxiety disorder animal model and a method for screening a preventive and therapeutic agent for treating anxiety disorder using the animal model. The mouse model for anxiety disorder of the present invention has normal learning ability, long-term memory and living activity but has a severe defect in memory extinction, therefore it can be effectively used for the screening of a therapeutic agent for anxiety disorder.

Description

[DESCRIPTION]

[invention Title]

PLC BETA 4 MUTANT MICE AS A MODEL FOR TESTING ANXIETY DISORDER DRUGS

[Technical Field]

The present invention relates to a use of a PLCβ4

(Phospholipaseβ4 ) knock-out animal, more precisely, a use or a method of using a PLCβ4 knock-out animal as an anxiety disorder animal model and a method for screening a preventive and therapeutic agent for treating anxiety disorder using the animal model.

[Background Art] Anxiety disorder includes phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder. Phobic disorder is the state of feeling fear for specific object or under specific circumstance and therefore avoiding constantly the object or the circumstance. Social phobia, acrophobia, claustrophobia and expectation anxiety are all included in phobic disorder. Generalized anxiety disorder is the state of constant anxiety in every day life widely and persistently. Obsessive compulsive disorder is a problem of repeating specific behavior or thought regardless of one's will. Compulsive idea or anankastic behavior might be normal to some degree, but when the compulsive idea or anankastic behavior disturbs every day life or annoys mind and body, it is diagnosed as obsessive compulsive disorder. In this case, anankastic personality such as accuracy, perfectionism, and principlism is shown as well. Post-traumatic stress disorder is a problem or illness that someone thinks or dreams or re-experiences a horrible accident that he or she had been through but not every common people could experience generally such as war, airplane crash, rape, collapse (Sampoong department collapse) and thereby the person becomes insensible to the outside world and exhibits abnormal symptoms of autonomic nervous system. Somatoform disorder exhibits physical symptoms which might be the signs of a disease, but in reality, most of the physical symptoms at this time have nothing to do with a disease but caused by a mental conflict. Sometimes, a mental conflict or a psychological factor can cause real physical symptoms or diseases, which are called psycho-physiological disorders. In most cases, when a mental conflict is expressed via physical symptoms, it can be done unconsciously. Therefore, it is quite different from malingering, the physical symptoms pretended by a patient deliberately. It is rarely expressed as dissociative disorder, which is the state of losing a part of mental functions temporarily like in ^λDr. Jekyll and Mr. Hyde' .

Mediodorsal thalamus (MD) is a part of limbic system, which is linked to prefrontal cortex involved in emotion, amygdala and hippocampus one another. However, the mechanism of MD in relation to emotional act has not been disclosed, yet. Phospholipase β4 (PLCβ4) is largely expressed in MD, but hardly expressed in prefrontal cortex or amygdala playing an important role in fear extinction

(Vertes RP. Neuroscience, 2006 Sep 29; 142 (1 ): 1-20; Oyoshi

T, et al._r J Neurosci. 1996 Sep 15; 16 (18 ): 5812-29; Kuroda

M, et al., Prog Neurobiol . 1998 Mar; 54 (4 ): 417-58 ; Nakamura M, et al., Eur J Neurosci. 2004 Dec; 20 (11 ): 2929- 44) .

Therefore, the present inventors studied and confirmed that PLCβ4 knock-out mice exhibited serious defect of memory extinction , so that it could be used as an anxiety disorder animal model, leading to the completion of this invention.

[Disclosure] [Technical Problem] It is an object of the present invention to provide a

PLCβ4 (Phospholipaseβ4 ) knock-out animal model demonstrating severe defect of memory extinction that is useful for the screening of a preventive or therapeutic agent for anxiety disorder.

[Technical Solution]

To achieve the above object, the present invention provides a method of using the PLCβ4 knock-out animal exhibiting severe detect of memory extinction as an anxiety disorder animal model.

The present invention also provides a use of the PLCβ4 knock-out animal demonstrating defect of memory extinction as an anxiety disorder animal model. The present invention further provides a method of screening a preventive and therapeutic agent for mental disorder using the PLCβ4 knock-out animal.

Hereinafter, the present invention is described in detail.

The present invention provides a use and a method of using the PLCβ4 knock-out animal demonstrating defect of memory extinction as an anxiety disorder animal model and a method for screening a preventive and therapeutic agent for mental disorder using the PLCβ4 knock-out animal.

The above method is composed of the following steps:

1) administering candidates for the preventive and therapeutic agent for anxiety disorder to the PLCβ4 knock- out animals:

2) testing memory extinction of the animals treated with the candidates of step 1) : and

3) selecting those candidates which recovered memory extinction significantly, by comparing with the control group not treated with the candidates.

In step 1) , the anxiety disorder is preferably selected from the group consisting of phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder, but not always limited thereto.

In step 1) , the animals are mammals, preferably mice, rats, pigs or monkeys, and more preferably the mice developed from the embryo (Accession No: KCTC 11247BP) deposited by the present inventors previously, but not always limited thereto.

In step 1), the candidates can be selected from the group consisting of peptides, proteins, non-peptide compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts, and sera. These compounds can be either novel compounds or well- informed compounds. The candidate compound can form a salt. For example, the salts of the candidate compounds are physiologically acceptable acids (inorganic acids) or bases (organic acids) and a physiologically acceptable acid added salt is more preferred. The salts are exemplified by the salts of inorganic acids (ex. hydrochloric acid, phosphoric acid, hydrobromic acid and sulfuric acid) and organic acids (ex. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methyl sulfonic acid and benzene sulfonic acid) .

A method for administering the above candidate can be selected from the group consisting of oral administration, intravenous injection, hypodermic injection, intradermal administration, and intraperitoneal injection, considering the symptoms of a target animal and characteristics of the candidate. The dosage of the candidate can be determined by considering the administration method and the characteristics of the candidate.

In step 2), the memory extinction test is performed as follows: conditioning is first induced and 1 - 60 hours later conditioned environment is periodically exposed, followed by measuring freezing response. It is preferred to perform the conditioning by the following cycles: 20 - 30 times for 15 - 50 seconds at 3 - 180 seconds intervals, but not always limited thereto.

To construct the Phospholipase β4 (PLCβ4) knock-out mouse, the mouse deleted with PLCβ4 which is largely expressed in mediodorsal thalamus (MD) but hardly expressed in prefrontal cortex or amygdala playing an important role in fear extinction, the present inventors constructed PLCβ4 deleted vector and then introduced the vector into embryonic stem cells of a mouse. After culturing the PLCβ4 deleted embryonic stem cell clone, the clone was injected into blastocoel of the blastocyst, followed by mating with a male which was vesactomized. Then, the blastocyst was transplanted into the uterus of a surrogate mother mouse to induce the development of a chimera mouse. The chimera mouse was mated with a wild-type mouse to generate a heterozygote mouse having the genotype of PLCβ4 +/-. The female and male heterozygote mice were mated each other to generate a mutant mouse having the genotype of PLCβ4-/- (PLCβ4 knock-out mouse) . In this invention, the PLCβ4 knock-out mouse constructed in the above was used as an anxiety disorder animal model and the embryo of the PLCβ4 knock-out mouse was deposited at KCTC (Korean Collection for Type Cultures) on December 6, 2007 (Accession No: KCTC 11247BP) . The present inventors investigated learning ability, long-term memory, living activity and memory extinction of the PLCβ4 knock-out mouse by measuring freezing response in an extinction chamber where outside noise is blocked, in order to judge whether or not this mouse could be used as an anxiety disorder model.

In the learning ability test as one of the typical fear conditioning test, when conditioning attempt to a specific sound was increased, freezing response (%) of the PLCβ4 knock-out mouse was also increased (see Figure 1) , suggesting that leaning ability of the PLCβ4 knock-out mouse was normal.

In the long term memory test of the PLCβ4 knock-out mouse, the rate of freezing response (%) of the knock-out mouse to the conditioned sound was similar to that of a wild-type mouse (see Figure 2 and Figure 3), suggesting that long term memory of the PLCβ4 knock-out mouse was normal .

In the living activity test, the distance that the PLCβ4 knock-out mouse moved in the extinction chamber where outside noise was blocked was measured. The result was not much different from that of a wild-type mouse (see

Figure 4), suggesting that living activity of the PLCβ4 knock-out mouse was normal not to induce freezing response.

In the memory extinction test, after 24 hours of conditioned learning of a specific sound, the wild-type mouse lost fear memory of the sound after 20 times of trials, while the PLCβ4 knock-out mouse did not lose the memory (see Figure 5) . Another 24 hours later, the wild- type mouse lost fear memory of the sound after 6 times of trials, whereas the PLCβ4 knock-out mouse still had the memory (see Figure 6) . So, it was proved that the PLCβ4 knock-out mouse had abnormal fear memory extinction to a specific sound.

In general, anxiety disorder patients are treated by exposing them repeatedly on the fear conditioned environment which is not dangerous, though. This method has been used for a long time. The above fear-conditioned memory extinction test performed in this invention using the animal having most human like effect has long been used since Pavlov (1927) (Myers KM and Davis M. Neuron. 2002 Nov 14;36 (4) :567-84; Milad MR and Quirk GJ. Nature. 2002 Nov 7;420 (6911) :70-4; Marsicano G, et al., Nature. 2002 Aug 1;418 (6897) :530-4) .

As explained hereinbefore, the PLCβ4 knock-out mouse exhibited normal learning ability, long-term memory and living activity, but had significantly reduced fear-memory extinction, so that it could be used effectively as an anxiety disorder animal model.

[Advantageous Effect] The PLCβ4 knock-out mouse of the present invention had normal leaning ability, long-term memory and living activity, but had significantly reduced fear-memory extinction . So, this mouse can be effectively used as an animal model for the screening of a preventive and therapeutic agent for various anxiety disorders.

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a graph illustrating the learning ability of the PLCβ4 knock-out mouse in the classical fear conditioning test:

+/+: wild-type mouse; and -/-: PLCβ4 knock-out mouse.

Figure 2 is a graph illustrating the long-term memory of the PLCβ4 knock-out mouse to a specific conditioned sound, investigated 24 hours later: +/+: wild-type mouse; and -/-: PLCβ4 knock-out mouse.

Figure 3 is a graph illustrating the long-term memory of the PLCβ4 knock-out mouse to a specific conditioned sound, investigated 48 hours later: +/+: wild-type mouse; and

-/-: PLCβ4 knock-out mouse.

Figure 4 is a graph illustrating the distance that the PLCβ4 knock-out mouse moved in the test chamber for memory extinction to a specific conditioned sound:

+/+: wild-type mouse; and

-/-: PLCβ4 knock-out mouse.

Figure 5 is a graph illustrating the result of fear memory extinction test to a specific conditioned sound (extinction training day 1) with the PLCβ4 knock-out mouse in the test chamber for memory extinction to a specific conditioned sound:

+/+: wild-type mouse; and

-/-: PLCβ4 knock-out mouse. Figure 6 is a graph illustrating the result of memory extinction recall test to a specific conditioned sound (recall of extinction day2) with the PLCβ4 knock-out mouse in the test chamber for memory extinction to a specific conditioned sound, measured after 24 hours from the fear memory extinction learning:

+/+: wild-type mouse; and

-/-: PLCβ4 knock-out mouse.

[Mode for Invention] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Construction of Phospholipaseβ4 (PLCβ4) knockout mouse <!-!> Construction of PLCβ4 deleted vector

To separate PLCβ4 gene from mouse genome, the certain region of PLCβ4 cDNA (SEQ. ID. NO: 1) ranging from the starting codon to 226-369^th nucleotides was isolated by RT- PCR, which was used as a probe for hybridization to 129/vsJae mouse genome DNA library (LamdaFixII, Stratagene Inc., USA). After selecting genomic clone phage having PLCβ4 gene, it was examined whether or not the gene was PLCβ4 by using restriction enzyme map, Southern blotting and nucleotide sequencing. To construct PLCβ4 deleted vector, a part of X domain of PLCβ4 protein was eliminated from the PLCβ4 gene clone, which was cloned into PSK-plasmid vector (Stratagene Inc., USA) . To increase targeting efficiency, thymidine kinase gene cassette and negative selection marker were inserted into the 3' homologous fragment of the targeting vector containing deleted PLCβ4 gene.

<l-2> Cell culture

Jl embryonic stem cells were used as a host cell line for the transfection with the above targeting vector constructed in Example <1-1>. Jl embryonic stem cells (provided by R. Jeanisch, MIT, USA) were inoculated into ES medium prepared by supplementing 15% fetal bovine serum (Hyclone Co., USA), 1* penicillin-streptomycin, 1* non- essential amino acids (Gibco Co., USA), and 0.1 mM 2- mercaptoethanol to DMEM (Gibco Co., USA), followed by culture at 37 ^°C for 2 -3 days. The embryonic cells prepared from the culture were treated with 1 mM EDTA solution containing 0.25% trypsin to separate as single cells.

<l-3> introduction of PLCβ4 deleted vector into cells

Electroporation was performed to transfect the embryonic stem cells separated as single cells in Example <l-2> with the targeting vector constructed in Example <1- 1>. Particularly, the embryonic stem cells diluted at the concentration of 2^χlO⁷ cells/m# were mixed with 25 βg of the target vector DNA prepared in Example <1-1>, followed by electric shock at 270 V/500 μF. The embryonic stem cells were cultured in ES medium supplemented with 0.3 mg/m£ of G418 and 2 μM of ganciclovir for 5 - 7 days. The embryonic stem cell clone where PLCβ4 gene was precisely targeted by the targeting vector by homologous recombination was selected by Southern blotting.

<l-4> Generation of PLCβ4+/- mouse

To generate a chimera mouse having the genotype of PLCβ4 +/-, the embryonic stem cell clone selected in Example <l-3> was micro-injected into the fertilized blastocyst.

Particularly, C57BL/6J female and male mice (Jackson Laboratory, USA) were mated. The 3.5 p.c. female mouse was sacrificed by cervical dislocation. The uterus was extracted from the sacrificed female mouse and the bottom of the uterus was cut by scissors. 1 ml of injection solution comprising 20 mM HEPES, 10% fetal calf serum, 0.1 mM 2-mercaptoethanol and DMEM was refluxed using a 1 ml syringe. The blastocyst was separated from the uterus tissues by using a micro glass tube under the dissecting microscope. The separated blastocyst was placed in a drop of the injection solution pre-dropped on a 35 mm Petri-dish, which was used for the following experiment.

To introduce the embryonic stem cell clone selected in Example <l-3> into the separated blastocyst, 10 - 15 embryonic stem cell clones were sucked in an injecting pipette, which was inserted into blastocoel of the blastocyst with giving negative pressure to the direction of inner cell mass of the blastocyst by using a holding pipette and then the embryonic stem cell clones were injected into blastocoel of the blastocyst with a microinjector (Zeiss Inc., USA) by changing the pressure to positive. The blastocyst harboring the clones was mated with the vesactomized male mouse, which was transplanted into the uterus of the 2.5 p.c. pseudopregnant surrogate mother mouse to induce a chimera mouse, a kind of crossbred hybrid, from the embryonic stem cell clone (Jl) and the blastocyst of a C57BL/6J mouse. At this time, for the transplantation into the uterus, the abdomen of the surrogate mother anesthetized with avertine (1 mg/kg) was cut 1 cm; the upper part of the uterus was pulled about 2 cm up to the outside of the body with a pincette; a hole was made by an injection needle, and through this hole a micro glass tube was inserted, through which the blastocyst was injected; peritoneal membrane was sewed two stitches with a suture; and the outer skin was sealed with a medical clip. The blastocyst inserted with the embryonic stem cell clone was transplanted into the uterus of the surrogate mother mouse, followed by culture for approximately 19 days, leading to the fusion of embryonic stem cell originated cells and blastocyst originated cells, resulting in the construction of a chimera mouse having the genotype of PLCβ4 +/-.

<l-5> Generation of PLCβ4 knock-out mouse Each chimera mouse was mated respectively with the C57BL/6J and 129sv mice more than 20 times, from which C57BL/6J-PLCβ4+/- and 129sv-PLCβ4+/- mice were generated. The produced mice were mated each other to generate Fl generations ^ΛPLCβ4+/+ and PLCβ4-/-' , which were used for the following experiments. The genotype was confirmed by PCR. Primers used for the PCR were Kl (5'- CTCCACACTCTGCAACCTAC-3 ' ; SEQ. ID. NO: 2), K9 (5¹- AGTTACTTCTGGATTTTCAGCC-S' ; SEQ. ID. NO: 3) and PFK22 (5¹- CTGACTAGGGGAGGAGTAGAAG-3' ; SEQ. ID. NO: 4) and PCR was performed as follows: 94^°C for 30 seconds, 58^°C for 30 seconds, and 72^°C for 30 seconds (40 cycles) . Kl and K9 primers were the primer set to confirm the genotype of the normal mouse and Kl and PFK22 were the primer set to confirm the genotype of the mutant mouse. Bands corresponding to each PCR product were confirmed on 1.5% EtBr/aragose gel (PLCβ4+/+: 190 bp, PLCβ4+/-: 250/190 bp and PLCβ4-/-: 250 bp) .

Example 2: Mouse raising and organization The mice were raised in SPF (specific pathogen free) environment where temperature was maintained at 22 ^°C and humidity was regulated at 55% and water and feed were provided freely under the light cycle of 12 hour light/12 hour dark. The mice for the experiment were all male and 6 - 18 mice in total. T-test and repeated two-way ANOVA test were performed for the statistical treatment.

Example 3: Investigation of learning ability of the PLCβ4 knock-out mouse under the classical fear condition Freezing response was measured to investigate learning ability of the PLCβ4 knock-out mouse.

In the conditioning chamber shutting out the outside sound, the mice were forced to listen to a specific unusual sound (2900 Hz, 100 db) for 30 seconds. Electric shock (0.5 mA) was given to the sole of the foot for 1 second, which was the cause of freezing response. The shock was given at 120 seconds intervals three times and it was arranged to end the shock at the same time with the end of sound. As a result, through the above learning, in the first trial, freezing response was 0%, but as conditioning went on, trial by trial, freezing response to the sound was increased in both wild-type mice and the PLCβ4 knock-out mice (Figure 1) . That is, there was no difference in leaning ability between the wild-type mice and the PLCβ4 knock-out mice.

Example 4: Investigation of long-term memory of the PLCβ4 knock-out mouse Freezing response was measured to investigate long- term memory of the PLCβ4 knock-out mouse.

In a different chamber having totally different conditioning (different sound, smell, light, color, texture, etc.), sound-conditioning was induced to investigate memory for the specific sound. After 24 and 48 hours, the sound was given once again for 30 seconds and then freezing response was measured.

As a result, the PLCβ4 knock-out mouse exhibited freezing response of 93.6 ± 0.8% [mean ± SEM (standard error of the mean), Figure 2] and 95 ± 1.1% (Figure 3), while the wild-type mouse exhibited freezing response similarly of 88.3 ± 2.8% (Figure 2) and 84.4 ± 2.9%

(Figure 3) . So, long term memory for a specific sound of the PLCβ4 knock-out mouse was normal.

Example 5: Investigation of living activity of the PLCβ4 knock-out mouse

Freezing response is related to living activity of a mouse, so the distance that the mouse moved, which is the index for living activity, was measured. 24 hours after conditioning, the distance that the mouse moved for 10 minutes in the same chamber as described in Example 4 was measured.

As a result, the gap between the total distance that the PLCβ4 knock-out mouse moved (2854.2 ± 150 cm) and the total distance that the normal mouse moved (2394.3 ± 230.5 cm) was not big (Figure 4), suggesting that living activity of the PLCβ4 knock-out mouse was similar to that of the wild-type mouse.

Example 6: Investigation of memory extinction of the PLCβ4 knock-out mouse

Sound conditioning was induced in both wild-type mice and the PLCβ4 knock-out mice by the same manner as described in Example 3. 24 hours later, the mice listened to the sound (30 seconds) 20 times in the conditioned sound memory test chamber (extinction day 1) . The intervals between the sounds were set at 5 seconds. 24 hours later, the mice listened to the sound again 6 times (extinction day 2 ) .

As a result, the wild-type mice demonstrated that their fear memory of the sound conditioned by 20 times of trials was extinct, whereas the PLCβ4 knock-out mice did not lost their memory (Figure 5) . 48 hours after the sound conditioning, the wild-type mice demonstrated that their fear memory against the sound was extinct after 6 trials, while the PLCβ4 knock-out mice still exhibited significant freezing response (Figure 6) .

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS]

[Claim l]

A method of using a phospholipaseβ4 knock-out animal having defect in memory extinction as an anxiety disorder animal model.

[Claim 2]

A use of the phospholipaseβ4 knock-out animal having defect in memory extinction as an anxiety disorder animal model.

[Claim 3]

The use or the method according to claim 1 or claim 2, wherein the anxiety disorder is selected from the group consisting of phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder.

[Claim 4]

The use or the method according to claim 1 or claim 2, wherein the animal of step 1) is a mammal.

[Claim 5] The use or the method according to claim 1 or claim 2, wherein the animal of step 1) is a mouse, a rat, a pig or a monkey.

[Claim 6] The use or the method according to claim 1 or claim 2, wherein the animal of step 1) is the mouse developed from the embryo deposited at KCTC (Accession No: KCTC 11247BP) .

[Claim 7] A method for screening a preventive and therapeutic agent for anxiety disorder comprising the following steps:

1) administering candidates for a preventive and therapeutic agent for anxiety disorder to the PLCβ4 knockout animals: 2) testing memory extinction of the animals treated with the candidates of step 1) : and

[Claim 8]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the anxiety disorder is selected from the group consisting of phobic disorder, generalized anxiety disorder, obsessive compulsive disorder, post-traumatic stress disorder, somatoform disorder, dissociative disorder and factitious disorder.

[Claim 9]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the animal of step 1) is a mammal.

[Claim lθ]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the animal of step 1) is a mouse, a rat, a pig or a monkey.

[Claim 11]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the animal of step 1) is the mouse developed from the embryo deposited at KCTC (Accession No: KCTC 11247BP) .

[Claim 12]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the candidate of step 1) is selected from the group consisting of peptide, protein, non-peptide compound, synthetic compound, fermented product, cell extract, plant extract, animal tissue extract and serum.

[Claim 13] The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 7, wherein the testing of memory extinction of step 2) is performed after 1 - 60 hours from the conditioning and after exposing on the conditioned environment, followed by measuring freezing response.

[Claim 14]

The method for screening a preventive and therapeutic agent for anxiety disorder according to claim 13, wherein the conditioning is performed by the following cycles: 20 - 30 times for 15 - 50 seconds at 3 - 180 seconds intervals.