WO2004064854A1

WO2004064854A1 - Method for enhancing learning and memory by suppressing the activity of ncx2 protein

Info

Publication number: WO2004064854A1
Application number: PCT/KR2003/000280
Authority: WO
Inventors: Hee-Sup Shin; Daejong Jeon; Hyewhon Rhim; Yu-Mi Yang
Original assignee: Korea Institute Of Science And Technology
Priority date: 2003-01-17
Filing date: 2003-02-08
Publication date: 2004-08-05
Also published as: AU2003211293A1; KR20040066248A; KR100514090B1

Abstract

The present invention relates to a method for enhancing learning and memory, particularly relates to the method for enhancing learning and memory by suppressing the activity of NCX2 protein. As the transgenic mouse defected with NCX2 gene showed enhanced learning and memory compared with a normal mouse, the substance suppressing the activity of NCX2 protein can effectively be used as a promoter of learning and memory, and NCX2 gene and its protein can be used to screen the substance for enhancing learning and memory.

Description

METHOD FOR ENHANCING LEARNING AND MEMORY BY SUPPRESSING THE ACTIVITY OF NCX2 PROTEIN

FIELD OF THE INVENTION

The present invention relates to a method for enhancing learning and memory, particularly relates to a method for enhancing learning and memory by suppressing the activity of NCX2 protein.

BACKGROUND ART OF THE INVENTION

The physiological function of NCX (Sodium-Calcium Exchanger) is to exchange three Na⁺ for one Ca²⁺ electrochemically by the generation of electrochemical gradient running along plasma membrane. In general, NCX discharges Ca²⁺ depending on electrochemical Na⁺ gradient. However, it has also been shown that Ca²⁺ influx through the NCX can occur under certain conditions (Hryshko and Philipson, 1997, Basic Res . Cardiol . , 92, 45-51; Blaustein and Lederer, 1999, Physiol . Rev. , 79, 763-854). Despite the importance of Ca²⁺ homeostasis, the physiological role of NCX in the brain is not well understood.

NCX and plasma membrane calcium-ATPase (PMCA) regulate intracellular Ca²⁺ concentration ([Ca²⁺]i) in excitatory cells such as nerve cells and heart muscle cells in the resting stage. PMCA binds Ca²⁺ with a high affinity but with a relatively low turnover rate (i.e., the amount of Ca²⁺ transported per carrier per unit time, typically ~100 s^-1) . In contrast, NCX has about a 10-fold lower affinity but a 10- to 50-fold higher turnover rate for Ca²⁺ compared to PMCA. When cells are activated and [Ca²⁺]i is increased, PMCA is saturated with Ca²⁺, resulting in the limitation of discharging Ca²⁺. Then, NCX can play a superior role in such condition (Sanchez-Armass and Blaustein, 1987, Am . J. Physiol . , 252, C595-603; Blaustein and Lederer, 1999, Physiol . Rev. , 79, 763-854). Three different isoforms of NCX (NCX1, NCX2,

NCX3) are encoded by distinct genes in mammals (Nicoll et al . , 1990, Science, 250, 562-565; Li et al . , 1994, J. Biol . Chem . , 269, 17434-17439; Nicoll et al . , 1996, J. Biol . Chem . , 271, 24914-24921). The expression of NCX2 in brain is 4-10 times higher than that of NCX1 and more than 100 times higher than that of NCX3 (Yu and Colvin, 1997, Brain Res . Mol . Brain Res . , 50, 285-292). Three different isoforms of NCX have similar characteristics (Linck et al . , 1998, Am . J. Physiol . , 274, C415-423; Blaustein and Lederer, 1999, Physiol . Rev. , 79, 763-854), but exhibit differences in expressions during development and in adults (Sakaue et al., 2000, Brain Res . , 881, 212-216; Gibney et al . , 2002, Neuroscience, 112, 65-73; Yu and Colvin, 1997, Brain Res . Mol . Brain Res . , 50, 285-292).

NCX was proved to contribute the elimination of intracellular Ca²⁺ in parallel nerve fiber (Regehr, 1997, Biophys . J. , 73, 2476-2488) and purkinje cells (Fierro et al . , 1998, J. Physiol . , 510, 499-512) of the cerebellum. In another study, an administration of antisense oligodeoxynucleotide to a conserved region of the three NCX isoforms resulted in a slower return to baseline Ca²⁺ levels following activation of Ca²⁺ influx by NMDA (N-methyl-D-aspartate) (Ranciat-McComb et al . , 2000, Neurosci . Lett . , 294, 13-16). It was also reported that NCX plays an important role in regulation of [Ca²⁺]i in the region of synapse of hippocampus nerve and affects synaptic vesicle recycling (Reuter and Porzig, 1995, Neuron , 15, 1077-1084; Bouron and Reuter, 1996, Neuron , 17, 969-978) . Therefore, blocking NCX function by replacement of extracellular Na⁺ with Li⁺ increased [Ca²⁺]i during and after stimulation in nerve terminals, resulting in faster initial rates of exocytosis of synaptic vesicles. The fast and prolonged increase of [Ca⁺]i in postsynaptic neuron is caused by the huge amount of neurotransmitter secreted in presynaptic terminus and by the delayed clearance of Ca²⁺ in postsynaptic region. Therefore, the delayed elimination of Ca⁺ can enforce the activation of synapse in pre- and postsynapse. However, it has not been reported whether NCX can play any role in the regulation of synaptic plasticity.

The present inventors have studied on a method to enhance leaning and memory, during which prepared transgenic mice defected with NCX2 gene, a major isoform of NCX, in brain and investigated the activation of synapse therein. The present inventors have completed this invention by confirming that it is possible to enhance learning and memory by suppressing

NCX2 activity. Precisely, when NCX2 is suppressed, the clearance of Ca²⁺ is delayed, causing enforced activity of synapse in pre- and post synapse, which enhances leaning and memory.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a method for enhancing learning and memory by suppressing the activity of NCX2 and a substance for enhancing learning and memory that contains an inhibitor of NCX2 activity as an effective ingredient.

It is another object of the present invention to provide a method for screening the substance enhancing learning and memory using NCX2 gene and its protein.

Further features of the present invention will appear hereinafter.

I . The present invention provides a method for enhancing learning and memory by suppressing the activity of NCX2 protein.

The ways to suppress the activity of NCX2 protein are suppressing the expression of NCX2 gene by targeting NCX2 gene using gene-targeting method, suppressing the expression of NCX2 gene by targeting NCX2 gene using antisense, lowering the amount of NCX protein by suppressing the expression of NCX gene, inhibiting the activity of NCX protein, suppressing transcription of NCX gene or suppressing translation of mRNA, but not always limited thereto. In the preferred embodiment of the present invention, the present inventors prepared NCX2-knockout mice by targeting NCX2 gene and used the same for further experiments.

NCX2 regulates the activity of synapse by taking part in Ca²⁺ concentration. Even though the recovery of [Ca²⁺]i to the basal level following the removal of glutamate was significantly slower in the mutant compared to wild type cells after Ca²⁺ influx was accelerated by glutamate, the [Ca²⁺]i in baseline and at peak were not much different (see FIG. 3B and 3C) . Therefore, NCX2 was confirmed to regulate the activity of synapse by engaging in recovery of baseline [Ca²⁺]i level .

In NCX2-knockout mice, fEPSPs (field excitatory postsynaptic potentials) were not changed in the input- output relation (see FIG. 4A) . Also, NMDA receptor- mediated field reaction was not changed (see FIG. 4D) , and the mutant mice exhibited enhanced shot-term plasticity (see FIG. 4E and 4F) . Together, these data indicate that NCX2 regulates the secretion of neurotransmitter, is related to the regulation of Ca²⁺ in presynaptic terminals and changes short-term placiticity.

LTP was also increased (see FIG. 5A) and synapse was enforced (see FIG. 5B) in NCX2-knockout mice.

Further, synapse was not constantly decreased (see FIG. 5D) . The mutant slices exhibited a strong tendency for developing enhanced LTP. These results are summarized as a plot of plasticity vs. stimulation frequency in Figure 5G. It is apparent that the frequency sensitivity of LTP and LTD induction in the CA1 region of the hippocampus is drastically shifted to lowered stimulus frequencies in NCX2 mutant mice.

In addition, the NCX2-knockout mice showed remarkably enhanced learning and memory, which was proved by Morris water maze test (see FIG. 6A - FIG. 6G) . In relation to the effect of memory on behavior, the transgenic mice showed increased long-term memory on fear (see FIG. 7A - FIG. 7C) . Also, the mutant mice have an enhanced performance in the object recognition memory task (see Fig. 7D) .

As shown above, suppressing the activity of NCX2 prolongs the time required for clearance of the increased [Ca²⁺]i induced by neuronal activation and biases synaptic plasticity towards increased STP and

LTP, thereby enhancing the animal's capacity for learning and memory that requires the function of the hippocampus .

π . The present invention provides a method for screening a substance regulating learning and memory using NCX2 gene and its protein.

The screening method of the present invention comprises the following steps: (1) Preparing transformant by transfecting host cells with a vector containing a NCX2 structural gene and a reporter gene;

(2) Culturing the above transformant and specimens together for screening; and (3) Measuring the expression of the reporter gene.

Learning and memory can be enhanced by suppressing the activity of NCX2, meaning a substance suppressing the activity of NCX2 itself can be a

/ substance enhancing learning and memory. Therefore, substances suppressing the activity of NCX2 can be screened by conventional methods for screening substances suppressing protein activity.

A conventional method to screen substances suppressing protein activity is as follows.

First, prepare transformant by transfecting host cells with a vector containing a NCX2 structural gene and a reporter gene. Culture the transformant together with specimens for screening and measure the expression of the reporter gene, resulting in the screening of substances suppressing the expression of NCX2. At this time, various expression vectors, promoters or reporter genes can be used for the experiment according to the host cells and experimental condition, and it is common knowledge to the people in this field that there is not limited to a specific one.

HI . The present invention provides a substance enhancing learning and memory by suppressing the activity of NCX2 protein selected by the above screening method.

As mentioned before, the NCX2-knockout mice showed enhanced learning and memory, so that the substances suppressing the activity of NCX could be used for the prevention of schizophrenia and for the enhancement of learning ability. The substance suppressing the activity of NCX2 can be a compound that suppresses the activity of NCX2 by binding specifically to NCX2, an antibody that binds specifically to NCX2, a substance that suppresses the transcription of NCX2 gene and a substance that inhibits the translation of mRNA transcribed from NCX2 gene, but the substance is not always limited thereto. Besides the above substances, every substance that can suppress the activity of NCX2 can be used.

The above substance suppressing the activity of NCX2 can be administered orally or parenterally and be used in general form of pharmaceutical formulation. The substance suppressing the activity of NCX2 can be prepared for oral or parenteral administration by mixing with generally-used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients. Solid formulations for oral administration are tablets, pill, dusting powders and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc with one or more halocidin. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are exemplified by suspensions, solutions, emulsions and syrups, and the above- mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.

Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc., in addition to the active compound or compounds. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerinated gelatin, etc.

The substance suppressing the activity of NCX2 of the present invention can be administered in both ways of single dose and multiple doses. Precisely, the total effective dosage can be administered as single dose by bolus or infusion that takes relatively short period to be absorbed and as multiple doses by fractionated treatment protocol. The effective dosage is determined in consideration of administration passage, treatment frequency, age and other conditions. Thus, the particular optimum dosage and mode of administration required for the active compounds can be determined by any expert in the field.

IV. The present invention provides NCX2-knockout mice in which specific region of NCX2 gene is targeted by gene-targeting method. That is, the present invention provides homozygote transgenic mice having NCX2 -/- genotype in which NCX2 protein is not expressed because of the targeting of NCX2 gene.

The present inventors have deposited the embryos of the transgenic mice having NCX2 +/- genotype at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on August 6, 2002 (Accession No: KCTC 10322BP) . The present inventors obtained transgenic mice having NCX2 -/- genotype by crossing mice both obtained from embryos having NCX2 +/- genotype.

The present invention also provides a preparation method for homozygote transgenic mice having NCX2 -/- genotype. The_ preparation method for the NCX2-knockout mice comprises the following steps:

(1) Introducing a targeting vector to NCX2 gene into mouse embryonic stem cells;

(2) Obtaining chimera mice by inserting the above embryonic stem cells in blastocoel of blastular staged embryo;

(3) Obtaining heterozygote mice having NCX2 +/- genotype by breeding normal mice with the above chimera mice; and (4) Obtaining homozygote transgenic mice having NCX2 -/- genotype by breeding female heterozygote mice with male heterozygote mice.

Particularly, prepared a targeting vector to NCX2 gene first. The targeting vector of the present invention includes two homologous fragments for NCX2 gene, NLS-Cre, PGK promoter, neo gene, PGK poly A and HSV thymidine kinase. Homologous recombination is occurred in two homologous fragments of the targeting vector and start codon of exon 1 is replaced with NLS- Cre, by which NCX2 gene is not expressed (see FIG. 1) .

Cultured embryonic stem cell clones in which NCX2 gene targeting was confirmed, and then prepared chimera mice by inserting those clones into blastocoel of blastular staged embryo. After breeding embryonic stem cell-inserted blastular staged egg with a male that got a vasectomy, transplanted thereof in the uterus of 2.5 p.c. surrogate mother mouse to induce the development of chimera mice. About 19 days later, produced chimera mice in which embryonic stem cell-originated cells and blastula staged egg-originated cells were together.

Obtained homozygote transgenic mice having NCX2 -/- genotype of the present invention by breeding female heterozygote mice having NCX2 +/- genotype with male heterozygote mice having the same. Confirmed that the homozygote transgenic mice had NCX2 -/- genotype by performing Southern blot and PCR, so did that NCX2 protein was not expressed in the transgenic mice by carrying out Western blot analysis (see FIG. 1A - FIG. ID) .

The present inventors have deposited the embryos of the transgenic mice having NCX2 +/- genotype at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on August 6, 2002 (Accession No: KCTC 10322BP) .

The present invention further provides a preparation method for homozygote transgenic mice having NCX2 -/- genotype, in which heterozygote transgenic mice having NCX2 +/- genotype was first obtained by transplanting the above embryos in surrogate mother mouse and the obtained male and female heterozygote transgenic mice were bred each other.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram showing the structure of wild-type NCX2 locus, targeting vector and disrupted gene locus,

■: targeted exons (exon 1 and exon 2 in order),

^→: PCR primer site, Underline of wild-type NCX2 gene locus : NCX2 probe for Southern blot,

NLS: nuclear localization sequence, Cre: ere recombinase, Neo: NEO cassette, TK: TK cassette,

B: BamΑI , E: EcoRI , H: Hindi11, N: IVcoI, P: Pstl, X: Xhol ,

FIG. IB is a set of photographs showing the results of Southern blot (upper part) and PCR (lower part) with tail tip genomic DNAs of wild type, NCX2 +/- and NCX2 -/- mice for genotyping,

+/+: Wild type mice, +/-: NCX2 +/- mice, -/-: NCX2 -/- mice

FIG. 1C is a photograph showing the result of Western blot with membrane fractions of wild type, NCX2 +/- and NCX2 -/- mice whole brain, which was performed to confirm the expression of NCX2 protein, +/+: Wild type mice, +/-: NCX2 +/- mice, -/-: NCX2 -/- mice,

FIG. ID is a set of photographs showing the normal gross morphology of the hippocampus of wild type (left, +/+) and NCX2 mutant mice (right, -/-) , FIG. 2A is a photograph showing the NCX2 expression in RNA extracted from brain and skeletal muscle of wild type mice by RT-PCR, M: Marker, Lane 1: Control (water),

Lane 2: DNase-treated RNA extracted from skeletal muscle of 1 day pup,

Lane 3: DNase-treated RNA extracted from skeletal muscle of 6 weeks adult, Lane 4: DNase-treated RNA extracted from brain of 1 day pup,

Lane 5: DNase-treated RNA extracted from brain of 6 weeks adult,

Lane 6: cDNA of skeletal muscle of 1 day pup, Lane 7: cDNA of skeletal muscle of 6 weeks adult,

Lane 8: cDNA of brain of 1 day pup,

Lane 9: cDNA of brain of 3 weeks adult,

Lane 10: cDNA of brain of 6 weeks adult,

Lane 11: Genomic DNA

FIG. 2B is a photograph showing the result of Western blot with membrane fractions of 5-week old wild type tissues,

Lane 1: Spinal cord, Lane 2: Heart, Lane 3: Brain, Lane 4: Lung, Lane 5: Liver, Lane 6: Kidney, Lane 7: Pancreas,

Lane 8: Spleen, Lane 9: Skeletal muscle

FIG. 2C is a photograph showing the Cre recombinase expression in wild type, NCX2 +/- and NCX2 -/- mice detected by Northern blot analysis,

+/+: Wild type mice, +/-: NCX2 +/- mice,

-/-: NCX2 -/- mice, <— : Cre recombinase RNA,

FIG. 2D is a photograph showing the X-gal stained coronal brain slice of a NCX2+/-/CAG-CAT-Z mouse,

Inset: A brain slice of cerebellum (40X magnification)

FIG. 3A (upper part) is a set of graphs showing that the forward exchange current is activated when Li⁺ containing solution is substituted with Na⁺ containing solution,

+/+: Wild type mice, -/-: NCX2 -/- mice,

FIG. 3A (lower part) is a graph showing the current amplitude at a holding potential of -40 mV in wild type (8 slices, 5 mice) and mutant (6 slices, 4 mice) mice, FIG. 3B is a graph showing the glutamate-induced [Ca²⁺]i changes in wild type (continuous line: +/+) and mutant (dotted line: -/-) mice,

Scale bar (_) : Treated glutamate for 10 seconds

FIG. 3C is a graph showing the [Ca²⁺]i Decay Kinetics (percent decrease from the peak of glutamate- induced [Ca²⁺]i) in wild type (16 cells, 4 mice) and mutant (17 cells, 4 mice) mice,

FIG. 4A is a graph showing the input-output plot of synaptic transmission between stimulation strength and the corresponding fEPSP slope,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 4B is a graph showing the slope of fEPSPs elicited by a given presynaptic fiber volley,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 4C is a graph showing the plot of synaptic transmission between stimulation strength and presynaptic fiber volley amplitude,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 4D is a graph showing the NMDA receptor mediated synaptic potential in the presence of CNQX(10 μM) and reduced Mg²⁺(0.1 mM) ,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 4E is a graph showing the PPF at 50 ms interval interpulse in wild type and mutant mice (scale bar: 1 mV, 10 ms) ,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 4F is a graph showing the PTP performed with

50 μM D-AP5 in wild type and mutant mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5A is a graph showing the LTP elicited by a single, 1 s 100 Hz train in mutant (12 slices, 10 mice) and wild type (9 slices, 8 mice) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5B is a graph showing the LTP elicited by a single, 2 s 50 Hz train in mutant (11 slices, 5 mice) and wild type (13 slices, 7 mice) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5C is a graph showing the LTP elicited by a single, 1.5 in 10 Hz train in mutant (12 slices, 7 mice) and wild type (10 slices, 7 mice) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5D is a graph showing the LTD elicited by a single, 15 min 1 Hz train in mutant (12 slices, 10 mice) and wild type (9 slices, 8 mice) mice,

• : Wild type mice (+/+) , O: NCX2 -/- mice Δ: 2 μM of D-AP5 treated NCX2 -/-

FIG. 5E is a graph showing the LTD elicited by a single, 30 min 0.5 Hz train in mutant (12 slices, 10 mice) and wild type (9 slices, 8 mice) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5F is a graph showing the LTD elicited by a single, 75 min 0.2 Hz train in mutant (7 slices, 5 mice) and wild type (6 slices, 5 mice) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 5G is a graph showing the summary of synaptic plasticity at different stimulation frequencies (scale bars: 1.5 mV, 10 ms) ,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 6A is a graph showing the mean escape latency per day cross days during 7 days training in wild type (n=12) and mutant (n=13) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 6B is a graph showing the normal swim speed of wild type and mutant mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 6C is a graph showing the visible platform task over three training sessions in wild type (n=ll) and mutant (n=10) mice,

•: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 6D is a graph showing the time spent for searching target quadrant at the first probe test after

3 day training,

T: Target quadrant, R: Adjacent right quadrant, L: Adjacent left quadrant, 0: Opposite quadrant, •: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 6E is a graph showing the crossing number for the target platform position at the first probe test after 3 day training,

FIG. 6F is a graph showing the time spent for searching target quadrant at the second probe test after 7 day training,

FIG. 6G is a graph showing the crossing number for the target platform position at the second probe test after 7 day training,

FIG. 7A is a graph showing the freezing behavior on the day of training in wild type (n=12) and mutant (n=12) mice, Solid line: CS(tone, 20 s) ,

▼ : US (foot shock, 2 s) , •: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 7B is a graph showing the contextual fear conditioning 24 hours after training, •: Wild type mice (+/+), O: NCX2 -/- mice

FIG. 7C is a graph showing the cued fear conditioning 24 hours after training, •: Wild type mice (+/+) , O: NCX2 -/- mice

FIG. 7D is a set of graphs showing the mean exploratory preference to a novel object during training (left) and during each retention time (right) in wild type (n=15) and mutant (n=16) mice.

•: Wild type mice (+/+), O: NCX2 -/- mice

EXAMPLES

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: Preparation of NCX2-knockout transgenic mice

<1-1> Construction of a targeting vector

In order to generate knockout mice defected with the NCX2 gene, the present inventors took advantage of gene-targeting method.

Particularly, the present inventors separated mouse NCX2 gene from mouse 129/Sv genome phage library (lambda FIXII, Staratagene) using cDNA probe pll (Li et al . , 1994, J. Biol . Chem . , 269, 17434-17439) composed of 255 bp of 5' region of rat cDNA. Obtained three clones containing two exons encoding amino acid residues 1-226 and 227-446 of NCX2, which showed 96% homology with rat NCX2. Named them exon 1 and exon 2. Confirmed three overlapping phage clones containing those two exons by restriction mapping, oligonucleotides hybridization and sequencing.

Prepared a targeting vector for homologous recombination using the above genome clones. Particularly, confirmed that exon 1 was located in start codon (ATG) having 1.7 kb of NLS-Cre of pxCANCre (NLS: nuclear localization sequence, Cre: cre recombinase) (Kanegae et al . , 1995, Nucleic Acids Res . , 23, 3816-3821) . The targeting vector of the present invention contains neo cassette (PGK (phosphoglycerate kinase) promoter-neo-PGK polyA) , a selection marker, and 7.8 kb homologous fragment having pPNT thymidine kinase (Herpes simplex virus thymidine kinase) (Tybulewicz et al., 1991, Cell , 65, 1153-1163). Exon 1 was substituted with NLS-Cre recombinase gene and neomycin resistant gene cassette for positive selection by homologous recombination and HSV thymidine kinase gene was included for negative selection (FIG. 1A) . NCX2 gene is not expressed by the loss of exon 1 caused by gene targeting, which is because exon 1 encodes most of 5 front membrane permeable domains and contains start codon (ATG) of NCX2 (Li et al . , 1994, J. Biol . Chem . , 269, 17434-17439). The start codon (ATG) can be substituted with NLS-Cre recombinase gene by decomposing the start codon region of NCX2 with Ncol, and Cre recombinase can be regulated by NCX2 promoter gene.

<l-2> Culture of embryonic stem cells

Jl embryonic stem cell (ES cell) line was used for the transfection with a targeting vector generated in the above example <1-1>. Embryonic stem cell culture and embryo manipulation were performed as described in the following reference (Kim et al . ,

Na ture, 1997, 389:290-293). Jl embryonic stem cells

(obtained from Dr. R. Jeanisch, MIT, USA; 1989, Na ture,

342, 435-438) were maintained in DMEM (Gibco Co.) supplemented with 15% fetal bovine serum (Hyclone Co.), lx penicillin-streptomycin (Gibco Co.) and lx non- essential amino acid (Gibco Co.) at 37 °C for 2-3 days. Single cells were obtained by treating the cells with 1 mM EDTA solution containing 0.25% trypsin.

<l-3> Transfection with a targeting vector

Targeting vector generated in the example <1-1> was transfected into the single cells obtained in the above example <l-2> by electroporation. Particularly, 80 βg of targeting vector DNA was added into embryonic stem cells. After mixing, electroporation was performed with 270 V/500 μ¥ . The cells were cultured in ES medium containing 0.3 mg/mi of G418 and 2 μM of gancyclovior for 5-7 days. Correctly targeted embryonic stem (ES) cell clones were selected by using homologous recombination method, and cultured thereof in ES medium for 18-22 hours. The cells were treated with trypsin to obtain single cells. Live cells were selected and used thereof for microinjection.

<l-4> Generation of chimera mice

In order to generate chimera mice having NCX2+/- genotype, embryonic stem cell clones selected in

Example <l-3> were microinjected into fertilized blastula of C57BL/6J mice. Particularly, female and male C57BL/6J mice (Jackson Laboratory, USA) were mated, and 3.5 days (3.5 p.c.) after mating, the female mouse was sacrificed by cervical dislocation. Uterus was removed from the sacrificed female mouse and terminal region of the uterus was cut with scissors. Using 1 m-β syringe, 1 md. of injection solution containing 20 mM HEPES, 10% FBS, 0.1 mM 2-mercaptoethanol and DMEM was circulated. Blastula was separated from the above uterus using microglasstube under the dissecting microscopy. The separated blastula was transferred into the drop of injection solution placed on 35 mm petridish.

In order to insert the embryonic stem cell clones selected in the above Example <l-3> into the blastula, adjusted inner cell mass direction of blastula to negative pressure with holding pipette using microinjector (Zeiss Co.), and then inserted syringe containing 10-15 embryonic stem cell clones into blastocoel of the blastula, after which changed the pressure into positive pressure, resulting in the insertion of embryonic stem cell clones into blastocoel of the blastula. After mating a female mouse having embryonic stem cell-inserted blastula with a male mouse having undergone vasectomy, transplantation was performed into a uterus of a 2.5 p.c. surrogate mother mouse to induce the development of chimera mice, a kind of hybrids generated from embryonic stem cell clones (Jl) and blastula of C57BL/6J mice. For the transplantation, anesthetized the surrogate mother with avertine (1 mg/kg-body weight) and excised the abdomen about 1 cm^'. Pulled the upper part of uterus out about 2 cm using a pincette, and then made a hole in the uterus with a needle. Inserted the blastula through the hole using a micro glass tube. Took two stitches in the peritoneal membrane with a suture, and then sutured the outer skin with a clip for internal medicine. Transplanted the blastula, in which embryonic stem cells were inserted by the above procedure, into the uterus of the surrogate mother mouse and raised for about 19 days, by which obtained chimera mice having NCX2 +/- genotype, which was resulted from fusion of embryonic stem cell originated cells and blastula originated cells.

<l-5> Generation of NCX2 +/- heterozygote mice

The present inventors prepared germline- transmitted Fl heterozygote (NCX2 +/-) mice by breeding the male chimera mice obtained in the above Example <1-

4> with female C57BL/6J mice. Gene typing was performed to select heterozygote mice having NCX2 +/- genotype among them. Particularly, performed PCR with genomic DNA extracted from the tails of the mice. Used Fl primer represented by SEQ. ID. No 1, Bl primer represented by SEQ. ID. No 2 and B2 primer represented by SEQ. ID. No 3 for the PCR. PCR was performed at the following cycles: 3 minutes at 95 ^°C; 45 cycles of 40 seconds at 94^°C, 40 seconds at 60^°C, 40 seconds at 72^°C; 5 minutes at 72^°C.

As a result, confirmed just one band (494 bp) in normal mice (+/+), and observed two bands (494 bp and 347 bp) in heterozygote mice (+/-) (lower part of FIG. IB) .

<l-β> Generation of NCX2 -/- homozygote mice

The present inventors prepared homozygote transgenic mice having NCX2 -/- genotype by mating male and female heterozygote mice having NCX2 +/- genotype, which were selected in Example <l-5>. In order to confirm whether the prepared homozygote transgenic mice had NCX2 -/- genotype, performed PCR using tail genomic

DNA with the same method as the above Example <l-5> and also performed Western blot to confirm that NCX2 protein was not expressed in the transgenic mice. <l-6-l> PCR (polymerase chain reaction)

After extracting genomic DNA from transgenic mouse tail, PCR was performed under the same conditions of the Example <l-5>, that is; 1 μi of genomic DNA was used as a template and the primers represented by SEQ.

ID. No 1, No 2 and No 3 were used.

As a result, 494 bp band amplified from normal NCX2 gene was detected in wild-type (+/+) mice, along with 494 bp band, 347 bp band was detected in heterozygote mice. Meanwhile, only 347 bp band was detected in homozygote mice, suggesting that the homozygote transgenic mice of the present invention had NCX2 -/- genotype (lower part of FIG. IB) .

<l-6-2> Western blot analysis

In order to confirm that NCX2 protein was not expressed in NCX2-knockout mice having NCX2 -/- genotype of the present invention, the present inventors performed Western blot analysis following the method of Kim et al . (Kim et al . , 2001, Neuron, 31, 35-

45) . Particularly, wild type and homogygote transgenic mice were sacrificed by cervical dislocation to isolate cerebrum. The isolated cerebrums were homogenized in cold lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, Protease inhibitor cocktail [Boehringer Mannheim], Calpain inhibitors I and II). After low speed centrifugation (1,000 x g, 5 minutes, 4°C), the supernatants were centrifuged (28,000 x g, 15 minutes, 4^°C) again to obtain crude membrane fractions. The crude membrane fractions were separated in SDS PAGE gels (8%) for 2 hours, and blotted to nitrocellulose membranes with 90 V for 2 hours. After blocking the membrane on TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% skim milk for 1 hour, treated with the primary antibody (anti-NCX2, 1:10,000) prepared according to the conventional method for 4 hours. Performed ECL (chemiluminescence; Amersham, UK) to detect out NCX2 protein using peroxidase-conjugated anti-rabbit IgG2 secondary antibody.

As a result, NCX2 protein was not detected in the transgenic mice having NCX2 -/- genotype, suggesting that NCX2 gene was eliminated (FIG. 1C) . The present inventors deposited the embryos of the transgenic mice having NCX2 +/- genotype at Gene Bank of Korea Research

Institute of Bioscience and Biotechnology on August 6, 2002 (Accession No: KCTC 10322BP) . <l-6-4> Morphological analysis of NCX2-knockout mouse brain

After confirming the normal growth of NCX2- knockout mice, the present inventors investigated morphological changes in the mouse brain. Particularly, cut the center of the 7-week old NCX2-knockout mouse brain (8 (M) , and stained thereof with cresyl violet. Beforehand, fixed the mouse in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde at 4^°C for 2 days. Put thereof in paraffin wax according to the standard method, and then made 8 m slices with a microtome. Removed paraffin and washed. Stored the slices in 90% alcohol at 37°C for 12 hours, after which stained with crystal violet for 2 hours .

As a result, any morphological change in major cytostructural compartments such as hippocampus, cerebellum, cortex, thalamus, basal ganglia and amygdala was not detected in the normally grown up

NCX2-knockout mice (FIG. ID) .

Example 2: Analysis of NCX2 expression

Although NCX2 has been known to be expressed in brain and skeletal muscles (Li et al . , 1994, J. Biol . Chem . , 269, 17434-17439), the expression in skeletal muscles is still in controversy (Nicoll et al . , 1996, J. Biol . Chem . , 271, 24914-24921). In order to analyze the NCX2 expression in skeletal muscles, CNS, brain and spinal cord, the present inventors performed RT-PCR with mRNA. Particularly, extracted total RNA from brains and skeletal muscles (upper femoral region of hind leg) of both young and adult mice using TRIzol reagent by following the manufacturer's direction (Gibco BRL) . Amplified cDNA by PCR using DyNAzymeTMII DNA polymerase (Finnzymes Inc.) along with the extracted RNA and RT-F1 primer represented by SEQ. ID. No 4 as well as RT-Bl primer represented by SEQ. ID. No 5 under the same condition in the above Example <l-5>. Isolated the amplified PCR product on 2% agarose gel. Checked any contamination by substituting cytoplasmic template with water in a control group. In order to exclude any influence of genomic DNA on the PCR product, induced reverse transcriptional reaction without reverse transcriptase in another control group. The two control groups were confirmed to be negative.

As a result, the NCX2 expression was not observed in skeletal muscles and negative control groups (H0 and DNase-treated RNA) , and was limited to brain and spinal cord (FIG. 2A) , which was the different result with the earlier reports saying NCX2 was expressed in skeletal muscles.

In order to make sure that NCX2 mRNA is not expressed in skeletal muscles, extracted total protein from brain and skeletal muscles (upper femoral region of hind leg) of both young and adult mice, followed by performing Western blot analysis with the same procedure as used in the above Example <l-6-3>.

As a result, it was confirmed that NCX2 protein was expressed only CNS, brain and spinal cord (FIG. 2B) and was not expressed in skeletal muscles, which was proved by RT-PCR and Western blot analysis.

Example 3: Analysis of Cre recombinase expression

It was difficult to confirm that NCX2 protein was not expressed in NCX2-knockout mice since there was no antiserum against NCX2 protein. Therefore, the present inventors confirmed the expression of NCX2 protein by analyzing the expression of Cre recombinase in a targeting vector prepared for defecting NCX2 gene. Particularly, performed Northern blot to confirm the transcriptive product of Cre recombinase. Extracted total RNA (20 μg) from brain using TRIzol reagent (Gibco) , which was electrophoresed on 1% agarose gel containing formaldehyde. Transferred thereof to nylon membrane in 10X SSC for 20 hours. After UV cross- linking, hybridized the nylon membrane with pxCANCre- originated [ α³²P] dCTP-labeled 1060 bp Pacl/Notl- digested fragment at 65^°C for 20 hours. Washed the nylon membrane with 2X SSC, 1% SDS for 10 minutes, and washed twice again with 0.2X SSC, 1% SDS at 65^°C for 15 minutes. Then, performed autoradiography.

As a result, observed about , 1.65 kb of Cre recombinase transcriptive product in heterozygote and homozygote transgenic mice but not in wild type mice (FIG. 2C) . Thus, it was confirmed that the transgenic mice were defected with NCX2.

After confirming that Cre recombinase transcriptive product was expressed in the NCX2- knockout mice, the present inventors also confirmed the NCX2 expressed part in brain. Mated NCX2 heterozygote transgenic mice with CAG-CAT-Z reporter mice, after which selected mice having CAG-CAT-Z reporter gene and

NCX2+/- genotype. Stained the mouse brain with X-gal .

Particularly, selected mice having CAG-CAT-Z reporter gene and NCX2+/- genotype after crossing NCX2 heterozygote transgenic mice with CAG-CAT-Z reporter mice (Sakai and Miyazaki, 1997, Biochem . Biophys . Res . Commun . , 237, 318-324). Fixed the brain of the selected mouse (NCX2+/—CAG-Δ-Z) with phosphate buffer (pH 7.4) containing 3.7% formaldehyde, followed by cutting (50-70 μm) . Washed thereof with PBS (phosphate buffered saline) three times and suspended thereof in LacZ staining solution (0.05 M phosphate buffer, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgC12, 1 mg/mA X-gal) at 37 °C for overnight (Sakai and Miyazaki, 1997, Biochem . Biophys . Res . Commun . , 237, 318-324). Compared the level of X-gal staining thereof with those of positive control (CAG-CAT-Z/CAG-Cre) and negative control (CAG- CAT-Z) . As a result, confirmed strong X-gal staining in NCX2+/-/CAG-CAT-Z mice (meaning NCX2-Cre-mediative recombination) , suggesting that NCX2-Cre was a functional protein (FIG. 2D) , and observed NCX2 all over the brain and strong staining in hippocampus (CA1, CA2, CA3, DG) , cerebellum (granule, Purkinje) and cerebral cortex.

Example 4: Analysis of Incx in whole plasma membrane

In order to measure the loss of Incx, the present inventors measured forward exchange current (Ca²⁺ out, Na⁺ in) with whole-cell visual patch-clamp method. Particularly, used hippocampus slices (250 μm) for the experiment and performed whole-cell visual patch-clamp recording according to the method of Fierro et al (Fierro et al . , 1998, J. Physiol . , 510, 499-512) with a slight modification. Clamped cells at -40 mV using whole-cell electrode (3-5 M, series resistance < 20 M) . Poured the prepared solution in which pH was adjusted to 7.4, using CsOH (3 mM NaCl, 114 mM CsCl, 9 mM EGTA, 9 mM HEPES, 1.8 mM MgCl₂, 4 mM MgATP, 0.3 mM Tris-GTP, 5.4 mM CaCl₂ (H₂CaEGTA was additionally added to measure 100 nM free Ca²⁺ at room temperature) ) in pipette. In order to avoid contamination by Na⁺/K⁺ pump and K⁺-dependent NCX (NCKX) activity (Tsoi et al . , 1998, J. Biol . Chem . , 273, 4155-4162), used TEA-C1 in exterior solution without K⁺. Performed the experiment using EPC-9 amplifier and Pulse/Pulsefit software (HEKA, Germany) at room temperature. Recorded signals on chart record (Gould, Cleveland, OH) and videocassette recording tape using digital data recorder (VR-10B

Instrutech digital data recorder, Elmont, NY) .

Represented all data by mean±SEM. Performed each test per fragment and applied all agents to application medium. Added 5 mM TEA (tetraethylammonium chloride), 1 μM TTX (tetrodotoxin) and 10 μM bicucullin to every solution.

As a result, Incx was activated when the solution containing 124 mM Na⁺ was substituted with Li⁺ exterior solution. The peak of amplified electric current was about 50% decreased in transgenic mice, comparing with wild type mice (Wild type (+/+): -193.39 + 21.86 pA, n=8; Transgenic (-/-): -84.45+13.19 pA, n=6, student's t-test, p<0.01) (FIG. 3A) . Therefore, it was confirmed that NCX2 influenced Incx in CA1 pyramidal neuron, while other NCX heteroforms supplied residual current.

Example 5: Measurement of intracellular Ca²⁺

In order to investigate the effect of Incx decreased in slices of hippocampus neuron of transgenic mice on [Ca²⁺]ι homeostasis, the present inventors analyzed intracellular Ca²⁺ image. Particularly, obtained hippocampus slices (400 μM) from 2-3 week old mice. Suspended the slices in ACSF (124 mM NaCl, 3.5 mM KC1, 1.25 mM NaH₂P0₄, 2 mM CaCl₂, 1.3 mM MgS0₄, 26 mM NaHC0₃ and 10 mM glucose, pH 7.4, Sigma) for 1 hour. Enzyme-treated thereof in Neurobasal-A medium (GibcoBRL, Grand Island, NY, USA) containing 0.1% papain (Roche) at 32 ^°C for 30 minutes. Cut CAl pyramidal region and crushed thereof 5 times with Pasteur pipette sterilized by flame. Plated thereof on cover slip coated with poly-L-lysine. Measured intracellular Ca²⁺ using fura-2 acetomethyl ester (Fura-2/AM; supplied by Molecular Probe) , a marker for intracellular Ca2+ in cell body

(Kim et al . , 2002, Biochem . Biophys . Res . Commun . , 296, 247-254). While culturing CAl neurons for 60 minutes, washed thereof with HEPES buffer (125 mM NaCl, 3 mM KCl, 10 mM HEPES, 2 mM CaCl₂, 10 mM glucose and 1.3 mM MgS0₄) three times. Treated with 250 μM of glutamate for 10 seconds, after which monitored the decrease of

[Ca²⁺]ι for 4-5 minutes. Repeated the experiment three times each.

As a result, as reported earlier (Kim et al . ,

2002, Biochem, ^'Biophys, Res, Commun, 296, 247-254),

[Ca²⁺]i was rapidly increased in the early stage and slowly recovered to the basal level (FIG. 3B) . The basal level (Wild type (+/+): 80.4±3.23 nM; Transgenic

(-/-): 80.9+ 4.24 nM) and the peak value (Wild type

(+/+): 330.67+19.54 nM; Transgenic (-/-): 307.57+

30.19 nM) of [Ca²⁺]ι was not much different between wild type cells (n=16, 4 mice) and transgenic cells (n=17, 4 mice) which were both activated by glutamate. However, the recovery speed of [Ca²⁺]ι to basal level was much slower in transgenic mice after removing glutamate than in wild type mice (FIG. 3C) . Obtained decay constant (τ) by making it meet with single exponential curve. The values of decay constant of transgenic cells ( τ =26.25 + 6.21 s) were about 40% higher than those of wild type cells (τ =9.96+ 1.14 s) . Based on the above results, it was confirmed that NCX2 plays a role in recovery of Ca²⁺ to the basal level in pyramidal neurons of hippocampus after depolarization as well as in other cells (Ranciat-McComb et al . , 2000, Neurosci . Lett . , 294, 13-16; Tang et al . , 2000, J. Neurochem . , 74, 702-710; Domotor et al . , 1999, J. Physiol . , 515, 147- 155; Fierro et al . , 1998, J. Physiol . , 510, 499-512; Sanchez-Armass and Blaustein, 1987, Am . J. Physiol . , 252, C595-603) .

Example 6: Transmission of basal synapses in NCX2- knockout mice

In order to confirm whether the basal synapses of NCX2-knockout mice functioned normally, the present inventors analyzed fEPSPs (field excitatory postsynaptic potentials) in CAl region of hippocampus by applying the stimulus of Schaffer collateral/commissural fibers to the mice. Particularly, prepared hippocampus slices from 7-8 week old mice in oxygen-treated cold artificial central nervous system fluid (ACSF; 124 mM NaCl, 3.5 mM KCl, 1.25 mM NaH₂P0, 2 mM CaCl₂, 1.3 mM MgS0₄, 26 mM NaHC0₃ and 10 mM glucose, pH 7.4). Left the slices between the surface of ACSF and air in a warm humid (32°C, 95% 0₂/5% C0₂) recording chamber for 1.5 hours. Located anode-stimulating electrode in stratum radiatum of CAl region and measured extracellular field potential in stratum radiatum using glass microelectrode (borosilicate glass, 3-5 megaresistance, 3 M NaCl) (Jun et al . , 1998, Learn Mem . , 5, 317-330). Detected out the test response at 0.033 Hz. As a result, the slope of fEPSP induced by given presynaptic fiber volley was not very different between wild type and transgenic mice (FIG. 4A, 4B and 4C) .

Measured fEPSPs mediated by NMDA receptor after adjusting the Mg²⁺ concentration of ACSF solution to

0.1 mM by adding CNQX, a AMPA receptor inhibitor, with the concentration of 10 μM to ACSF solution. As a result, there was not much difference in fEPSPs mediated by NMDA receptor between wild type and transgenic mice (FIG. 4D) . Therefore, it was confirmed that NCX2 defection did not affect the function of basal synapse.

Example 7: Changes of short-term plasticity in NCX2- knockout mice

Ca²⁺ has been known to be related to the regulation of synaptic transmission. Thus, the present inventors investigated the effect of NCX2 defection on two types of presynaptic short-term plasticity (STP) . Firstly, measured PPF (paired-pulse facilitation) to analyze potential elevation of neurotransmitter secreted by two closely connected spatial stimuli . The increase of the secretion was caused by Ca⁺ remaining in presynaptic terminal after getting the first stimulus (Regher et al . , 1994, J. Neurosci . , 14, 523- 537). Confirmed that PPF was remarkably increased in transgenic mice under 100 ms interval interpulse (30 ms interval: p<0.001; 50 ms interval: p<0.01; 70 ms interval: p<0.01, student's t-test) comparing with in wild type mice after observing that Ca²⁺ in transgenic mice was eliminated slowly (FIG. 4E) .

Secondly, investigated the presynaptic short-term plasticity resulted from PTP (posttetanic potentiation) , in other words, high frequency tetanus (100 Hz, 1 sec) .

Observed that transmission of single 100 Hz tetanus stimulus was accelerated (EPSP) to bring it down to basal line within 3 minutes in the presence of D-AP5 (50 μM) functioning to hinder NMDA receptor. Also observed accelerated higher PTP peak in transgenic mice

(-/-: 218.3+11.3%, ρ<0.001, student's t-test) than in wild type mice (+/+: 157.1+8.4%, p<0.001, student's t- test) (FIG. 4F) . Based on the above results, it was confirmed that two types of presynaptic short-term plasticity were increased in NCX2-knockout mice.

Example 8: Analysis of the changes of long-term plasticity

There is a possibility of changes in LTP (long- term potention) and in LTD (long-term depression) since the short-term plasticity was changed in NCX2-knockout mice. Thus, the present inventors investigated both- directed synaptic plasticity in the range of 0.5-100 Hz to analyze long-term plasticity. Particularly, regulated basal stimuli to the level of about 40% of the maximum level of inducing reaction for LTP experiment. For LTD experiment, used 3-4 week old mice. Determined synaptic input-output curve by calculating fEPSP slope and relevant fiber volley size in terms of graph. Tested PPF with pulse intervals of 30, 50, 70, 100, 200 and 500 msec. Measured NMDA receptor-mediated EPSP after shutting down AMPA/kainite receptor using 10 μM of CNQX in a few experiments, in which Mg2+ concentration was adjusted to 0.1 mM and remaining

EPSPs were completely blocked by 50 μM of D-AP5. Used different level of stimuli for making fiber volley and NMDA receptor-mediated EPSP slope plot. In the PTP test, applied single stimulus at 100 Hz with NMDA receptor antagonist (D-AP5, 50 μM) for 1 second. Applied agents onto application medium at least 30 minutes in advance. Represented data by mean±SEM.

As a result, LTP was highly increased 50 minutes after single stimulus at 100 Hz was given in transgenic mice (-/-: 253.5+8.2%, n=12, p<0.001, student's t- test) , comparing with wild type mice (+/+: 143 + 5.1%, n=9, p<0.001, student's t-test) (FIG. 5A) . Synaptic enhancement was observed 50 minutes after single stimulus at 50 Hz was given in transgenic mice, but plasticity was not decreased in transgenic mice (-/-: 220.1+25.1%, n=ll, p<0.001, student's t-test), compared with in wild type mice (+/+: 124.3 + 15.2%, n=13, p<0.001, student's t-test) (FIG. 5B) . When stimulus at 10 Hz was given, transgenic mice showed increase LTP 50 minutes later while wild type mice showed lower level of synaptic plasticity (Wild type

(+/+): 117.2 + 6.0%, n=l; Transgenic (-/-): 158.2 + 5.6%, n=12, p<0.001, student's t-test) (FIG. 5C) .

Secondary, measured LTP induced by low-fremitus stimulus (LFS, from 3-4 week old mice) . As Bear and Abraham reported (Bear and Abraham, 1996, Annu . Rev. Neurosci . , 19, 437-462), LFS did not induce LTD in adults, so that 3-4 week old mice were used for analyzing LTD. As a result, LTD was decreased by the stimulus at 1 Hz (15 min, 900 pulse) in wild type mice (+/+: at 50 minutes, 81.5 + 78.5%, n=9, p<0.001, student's t-test) while synaptic depression was not induced in transgenic mice (-/-: at 50 minutes, 124.1+ 6.6%, n=12, p<0.001, student's t-test) (FIG. 5D) . However, synaptic reaction in transgenic mice was slowly increased as time went by.

Lastly, measured LTD induced by stimulus at 0.5 Hz (30 min, 900 pulse) . As a result, LTD was not induced by stimulus at 0.5 Hz in transgenic mice (-/-: at 50 minutes, 122.7+9.8%, n=12, p<0.001, student's t test), compared with in wild type mice (+/+: at 50 minutes 72.5+5.4%, n=9, p<0.001, student's t-test) (FIG. 5E) . In the meantime, the results of measuring LTD induced by 0.2 Hz showed that LTD (clear LTD) was certainly induced in transgenic mice (-/-: at 120 minutes, 76.7+6.1%, n=7, p<0.001, student's t-test), compared with in wild type mice (+/+: at 120 minutes 95.1 + 4.5%, n=6, p<0.01, student's t-test) (FIG. 5F) .

As seen hereinbefore, it was proved that neuron of NCX2-knockout mice held high concentration of [Ca²⁺]i long after getting stimulus. High [Ca²⁺]i resulted from delayed clearance of Ca²⁺ prohibits LTD from being induced by 1 Hz stimulus in transgenic mouse slices.

Example 9: Enhanced learning and memory in NCX2- knockout mice

In order to confirm whether the increased LTP could enhance learning and memory in NCX2-knockout mice, the present inventors performed Morris water maze test, which is hippocampus-dependent execute method depending on the animal's capacity to learn and memorize the relation between long distance stimulus and hidden escape platform (Morris et al . , 1982, Na ture, 297, 701- 708) . Particularly, used 8-12 week old mice for Morris water maze test. The water maze apparatus was constituted of round pool (white plastic, 120 cm in diameter, 93 cm in height) containing 24-26^°C water and made opaque with non-toxic water soluble paint. The pool was set in the center of a room (2.5 x 2.5 m) and 4 ques were hung on each side of wall. Trained group 1 to find hidden platform (a circle 10 cm in diameter, located 1 cm beneath water) during 7 sessions (4 times trial/session/day), so did group 2 during 4 sessions. Let mice watch the wall at random. Made mice find the platform for 60 seconds and rest for 30 seconds. When mice could not find the platform within 60 seconds, stopped the mice and let them in platform for 30 seconds. Carried out transmission test 3 times. The first transmission test was performed with group 1 and 2 at the end of the third session, the second transmission test was performed with group 1 at the end of the second session and the third transmission test was performed with group 2 two weeks after the forth session. While performing transmission test, removed platform and let the mice swim in the pool for 60 seconds. Followed the traces of the mice with inf ared-sensitive camera (Advanced VP 2000) connected to tracker unit. Saved the traces, which were collected by software (HVS Water for windows software,

HVS IMAGE Ltd) . Analyzed the required time in quadrant and crossing-times of platform. Used other mice (group 3) for visual platform test and performed hidden platform test using the same water maze. But this time, there were two differences: 3 trials/session/day; black platform, which was moved each time.

Used 3 groups of mice. Trained group 1 mice for acquisition test from day 1 to day 4, trained group 2 for long-term memory from day 1 to day 4 and trained group 3 for visual test.

As a result, escape latency (F[6, 138]=30.29, p<0.0001, both-directed repeated ANOVA) implying learning effect was decreased in wild type (n=12) and in transgenic mice (n=13) during the training session for acquisition test (FIG. 6A) . However, the transgenic mice showed remarkably low escape latency during the training session, suggesting that the transgenic mice acquired learning faster than the wild type mice (F[l, 23]=7.67, p<0.01, both directed repeated ANOVA) . In addition, big differences were seen between the wild type and the transgenic mice on day 2 (p<0.05), day 3 (p<0.05) and day 5 (p<0.05) sessions, which were confirmed by Scheffe's test (post hoc test) . The enhanced learning in the transgenic mice was also confirmed by the first transmission test done 3 days later (FIG. 6D and 6E) . Both the wild type and the transgenic mice spent much time in target quadrant, comparing to other quadrants (F[3, 69]=31.99, p<0.0001, both-directed repeated ANOVA) (FIG. 6D) , and the frequency of the passing through location was outnumbered that of the crossway location (F[3, 69]=17.43, p<0.0001, both-directed repeated ANOVA). The transgenic mice spent much more time in target quadrant than the wild type mice (student's t-test, p<0.05) (FIG. 6D) , and showed much accurate memory about the location of platform, which was proved by measuring the frequency of passing through the platform

(FIG. 6E) . After 7 day training, the second transmission test was done, in which both the wild type mice and the transgenic mice were proved to have a strong preference for the target quadrant (FIG. 6F and 6G) .

Next, investigated long-term memory of the transgenic mice. Precisely, trained group 2 mice for 4 days, during which escape latency of both wild type and transgenic mice were same. Carried out the third transmission test after 2 weeks. As a result, escape latency was still same in both the wild type (n=ll) and the transgenic mice (n=10) (FIG. 6C) . From the above results, the present inventors could exclude the possibility that non-associative factors might affect learning and memory during water maze test.

Example 10: Fear condition analysis

In order to figure out that memory could be enhanced through other behaviors, the present inventors investigated contextual fear condition that was known to be induced by the activity of hippocampus and tonsil of cerebellum (Phillips and LeDoux, 1992, Behav. Neurosci . 106, 274-285). Animals learn fear by new environment or conditional stimulus (CS) like mild shock on food, especially when it paired with hateful un-conditional stimulus (US) . They show conditional immobility response that is characterized by immobility and shrink right after getting conditional stimulus. In rodents, lesion of hippocampus is limited to two forms of fear condition: one is non-specific cue (chamber contextual) that is sensitive to the lesion of hippocampus and the other is specific cue (situation) that is not sensitive to the lesion of hippocampus. Contextual condition depends on hippocampus, but cued condition depends on tonsil of cerebellum.

The present inventors used fear regulating shock chamber (19 X 20 X 33 cm) containing stainless steel grid (5 mm in diameter, 1 cm away from the bottom) , and active monitor (WinLinc Behavioral Experimental control software, Coulbourn Instruments) . In order to give contextual and cued fear, put mice (8-12 weeks old) in fear-conditioned chamber for 2 minutes, during which gave auditory conditional stimulus (CS, white noise) for 20 seconds. For the last 2 seconds, applied 0.5 rriA shock as un-conditional stimulus to floor grid. Performed the protocol once. Based on the pilot experiment, determined the intensity of stimulus and the frequency of training to get optimum effect of learning. In order to investigate suggested learning capacity, put animals to new surroundings (new chamber, smell, floor and visual hint) after training and left for 24 hours. Exposed the animals to tone for the last 3 minutes of the test. Investigated fear response by measuring the length of immobility response time with a stopwatch. Observed basal behavior in the new surroundings for 6 minutes and then gave sound CS for 1 minute. Measured both contextual and cued conditions in shock chamber during 24 hours after one time CS/US training.

As a result, the behaviors before and after training of both wild type (n=12) and transgenic (n=12) mice were not much different under contextual fear condition (FIG. 7A) . 24 hours later, returned the mice to the same shock chamber and observed. Both the wild type and the transgenic mice showed fear response but the transgenic mice became rapidly insensitive in that chamber, suggesting that the transgenic mice had enhanced long-term memory to contextual fear response compared with the wild type mice (F[l, 22] =15.37, p<0.0001, both-directed, repeated ANOVA) .

From the Scheffe's test (post hoc test), it was proved that there was a big difference in contextual fear response between the two genotypes during the first (p<0.001), the second (p<0.01), the third (p<0.05) and the fifth (p<0.05) session (FIG. 7B) . Meanwhile, there was no difference in cued fear response between wild type (n=12) and transgenic (n=12) mice (FIG. 7C) , suggesting that the enhanced memory of the NCX2-knockout mice depended only on hippocampus- dependent fear conditioning.

Example 11: Analysis of recognition on subjects

The present inventors investigated the recognition capacity of mice based on their ability to differentiate (Vnek and Rothblat, 1996, J. Nerurosci . , 16, 2780-2787) a new one from a familiar one depending on the action of hippocampus (Mansuy et al . , 1998, Cell, 92, 39-49; Tang et al . , 1999, Nature, 401, 63-69; Podhorna and Brown, 2002, Genes , Brain and Behavior, 1, 96-110) . Particularly, trained 31 mice (Wild type (+/+) : 1 hour, n=6; (+/+) : 24 hours, n=9; Transgenic (- /-) : 1 hour, n=7 ; (-/-): 24 hours, n=9) in an open container (40X40X40 cm) for 3 days respectively. During the training, left two subjects for 5 minutes in the container in order for the mice to see. A mouse was regarded to recognize the subjects when its head faced the subjects within 1-inch distance. After 1 or 24-hour retention, put two subjects in the container for 5 minutes, but this time, one of the two familiar subjects was replaced with a new one. Measured the time to recognize one of the two subjects or just a new one, based on which analyzed recognition memory.

As a result, there was no difference in recognizing time for two subjects between the wild type

(n=15) and the transgenic (n=16) mice (FIG. 7D left) .

During 1 hour retention, both the wild type (n=6) and the transgenic (n=7) mice reacted rather to a new subject than a familiar one (F[l, 11]=14.55, p<0.01, both-directed, repeated ANOVA) , but there was no difference in response between the wild type and the transgenic mice (F[l, 11]=0.01, p=0.96, both-directed, repeated ANOVA) . However, during 24-hour retention, the transgenic mice (n=9) reacted to a new subject faster than the wild type mice (n=9) (F[l, 16]=16.48, p<0.001, both-directed, repeated ANOVA, Scheffe's post hoc test, p<0.01), suggesting that the transgenic mice had much enhanced recognizing capacity.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present inventors prepared transgenic mice whose NCX2 gene was targeted and performed experiments using the transgenic mice. As a result, the present inventors confirmed that the suppression of NCX2 activity causes the delay of Ca²⁺ clearance and biases synaptic plasticity towards increased STP and LTP, resulting in the enhancement of learning and memory. That is, NCX2 plays an important role in synaptic plasticity by maintaining required

[Ca⁺]ι by regulating the increase and the decrease of

[Ca²⁺]ι in pre- and post-synapse and NCX2 mutation affects hippocampus-dependent learning and memory.

Therefore, NCX2 gene or its' protein can be effectively used for screening a substance regulating learning and memory.

BUDAPEST TREATY ON THE INTERNATIONAL HSCOONITIQN OP THE DEPOSIT OF MICROORGANISMS :FOR THE PURPOSE OP PATENT PROCEDURE

INTERNATIONAL FORM

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7,1

TO : SHIN, Hee-Sup

Korea Institute of Science and Technology,

#39-1, Ha olgok-doπg, Seongbuk-ku, Seoul 136-791,.

Republic of Korea

I , IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:

C57BL/6J embryo lacking NCX2^?

KCTC 10322BP

π . SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompanied by:

I x ] a scientific description

[ ] a proposed taxonomic designation

(Mark with a cross where applicable). m. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on August 06 2002.

IV. RECEIPT ^'OF REQUEST FOR CONVERSION

The microorganism identified under I above was^' received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on

V. INTERNATIONAL DEPOSITARY. AUTHORITY

Name: Korean Collection for Type .Cultures

Address: Korea Research Institute bf Bioscience and Biotechnology (KRIBΪ3)

#52, Oun-dong, Yusong-Ku, Taejon 305-333, .

Republic of Korea Date: August 29 2002

Foππ BP/4 (KCTC Fuπn 17) sole page

Claims

W a is claimed is

1. A method for enhancing learning and memory by suppressing the activity of NCX2 protein.

2. The method for enhancing learning and memory as set forth in claim 1, wherein the NCX2 protein is suppressed by suppressing the expression of NCX2 gene.

3. The method for enhancing learning and memory as set forth in claim 2, wherein the expression of NCX2 gene is suppressed by targeting NCX2 gene using gene-targeting method.

4. The method for enhancing learning and memory as set forth in claim 2, wherein the expression of NCX2 gene is suppressed by targeting the gene using antisense.

5. A method for screening a substance regulating learning and memory using NCX2 gene or protein.

6. The method for screening a substance regulating learning and memory as set forth in claim 5, wherein the method comprises the following steps:

(1) Preparing a transformant by transfecting host cells with a vector containing a NCX2 structural gene and a reporter gene;

(2) Culturing the above transformant and specimens together for screening; and

(3) Measuring the expression of the reporter gene.

7. A regulating agent for learning and memory containing a suppressor of NCX2 protein activity.

8. The regulating agent for learning and memory as set forth in claim 7, wherein the agent suppresses the activity of NCX2, which causes the acceleration of Ca²⁺ influx and the delay of Ca²⁺ clearance, resulting in the increase of synaptic transmission.

9. A transgenic mouse having NCX2 -/- genotype in which NCX2 protein is not expressed.

10. A preparation method for the transgenic mice of claim 9 comprising the following steps: (1) Introducing a targeting vector to NCX2 gene into mouse embryonic stem cells;

(3) Obtaining heterozygote mice having NCX2 +/- genotype by breeding normal mice with the above chimera mice; and

(4) Obtaining homozygote transgenic mice having NCX2 -/- genotype by breeding female heterozygote mice with male heterozygote (NCX2 +/-) mice.

11. The preparation method for the transgenic mice as set forth in claim 10, wherein the targeting vector includes two homologous fragments to NCX2 gene, neo cassette and thymidine kinase gene cassette.

12. An embryo of heterozygote transgenic mouse having NCX2 +/- genotype (Accession No: KCTC 10322BP) .

13. A preparation method for homozygote transgenic mice having NCX2 -/- genotype, in which heterozygote transgenic mice having NCX2 +/- genotype was first obtained by transplanting the embryo of claim 12 in a surrogate mother mouse and the obtained male and female heterozygote transgenic mice were bred each other.