JP2016000011A - Probe which visualizes synapse enhancement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid probe for visualizing increase and neogenesis of synapse which occur in living brain, to provide an expression vector and the like in which the nucleic acid probe is incorporated, and to provide a method for visualizing synapse enhancement.SOLUTION: The invention relates to a nucleic acid probe for visualizing synapse enhancement comprising (a) a nucleic acid having a base sequence in which a part or all of 193-936 nt is deleted from a nucleic acid consisting of a specific base sequence encoding an ionic channel type receptor anchoring protein PSD-95, or a nucleic acid hybridizing to the nucleic acid under stringent conditions; (b) a nucleic acid consisting of a specific base sequence encoding a dendritic target element (DTE), or a nucleic acid hybridizing to the nucleic acid under stringent conditions; and (c) a nucleic acid encoding a reporter protein inserted between the above (a) and (b).

Description

  The present invention relates to a gene cassette, a nucleic acid probe, a protein probe, an expression vector incorporating the nucleic acid probe, a transgenic non-human animal incorporating the nucleic acid probe, and synaptic enhancement for visualizing the increase and new generation of synapses occurring in the living brain. It is related with the method of visualizing.

  The vertebrate nervous system is composed of nerve cells and glial cells. Among these cells, the nerve cell is a cell composed of a cell body and two types of processes, a dendrite and an axon. After integration, it is transmitted to the axon, and then transmitted to post-synaptic cells such as other nerve cells, muscles, or glandular cells via the synapse at the nerve terminal at the end of the axon. Stimulations transmitted by nerve cells are broadly classified into those that excite post-synaptic cells and those that suppress excitation. When a cell membrane (post-synaptic membrane) possessed by a post-synaptic cell receives a certain level of stimulation, a depolarizing synaptic reaction (excitatory synaptic potential occurs, and excitation is transmitted to the post-synaptic cell. Nerve cells that transmit to post-synaptic cells are called excitatory neurons, and conversely, those that have the function of suppressing the excitability of post-synaptic cells are called inhibitory neurons, which act on the postsynaptic membrane. Induction potential is called inhibitory synaptic potential, and typical excitatory neurons include pyramidal cells in the cortical motor area and motor neurons in the spinal cord, and hippocampal baskets as inhibitory neurons. Cells, Purkinje cells of the cerebellar cortex, Renshaw cells of the spinal cord, etc. are known.

  Neurons of the central nervous system with different functions described above interact in complex ways to transmit various information, and analyzing their interactions and dynamics can be used to analyze various brain functions (for example, It is essential to clarify the mechanism of memory and learning. In order to achieve such an object, attempts have been made so far to identify and identify excitatory and inhibitory neurons in the central nervous system. As a specific classification method, for example, a method of staining with a marker specific to nerve cells can be mentioned. However, in this method, it is necessary to fix the tissue in order to perform staining with a marker such as an antibody, and the activity of nerve cells in the living body cannot be analyzed in real time. On the other hand, as a method for observing in vivo proteins in real time, a method of expressing a target protein in vivo as a fusion protein with a fluorescent protein has been developed. For example, SEP-GluA1 is known as a probe for visualizing synaptic enhancement, but it has a long half-life and expresses fluorescence for more than a month with a synapse once enhanced. It is said that it is impossible to label. Moreover, although this probe can visualize the synaptic enhancement, it cannot artificially manipulate the synapse.

  In the human cerebral cortex, a neural network in which a huge number of neurons communicate with each other through about 60 trillion synapses is constructed, of which about 80% of excitatory synapses form a microprojection structure called spine. In response to learning or experience (memory), spines change their form and size dramatically, and accordingly change their electrical transfer efficiency. Therefore, spines are considered as memory elements for brain function. To date, no technique has been developed to visualize specific spines that have been activated with the aim of tracking the dynamic behavior of synapses associated with brain function.

  The present inventors are involved in an anchoring protein (for example, PSD-95) and synaptic plasticity of an ion channel receptor having a property of being arranged and accumulated in a post-synaptic cell excited by a certain stimulus or more. Focusing on two proteins of dendritic target element (DTE) translated at the end of synapse, the fusion protein of these and reporter protein succeeded in visualizing the increase in synapse, leading to the completion of the present invention. It was.

That is, the present invention is as follows.
[1] (a) having a nucleotide sequence obtained by deleting a part or all of 192 nt to 935 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95 A nucleic acid, or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (b) a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3 encoding a dendrite target element (DTE), or the nucleic acid and a stringent A gene cassette for visualizing synaptic enhancement comprising a nucleic acid that hybridizes under mild conditions.
[2] The gene cassette according to [1], wherein the nucleic acid identified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193nt to 936nt of SEQ ID NO: 1 have been deleted. .
[3] (a) having a nucleotide sequence obtained by deleting a part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95 A nucleic acid or a nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above A nucleic acid probe for visualizing synaptic enhancement, comprising a nucleic acid encoding a reporter protein inserted between (b).
[4] The nucleic acid encoding the reporter protein is selected from the group consisting of Venus, green fluorescent protein, channelrhodopsin, halorhodopsin, glutamate decarboxylase, luciferase, chloramphenicol acetyltransferase, β-galactosidase, and β-glucuronidase. The nucleic acid probe according to [3] above, which is a nucleic acid encoding one or more selected proteins.
[5] The method according to [4] above, further comprising a nucleic acid encoding a protein selected from the group consisting of Rho family G protein, FAK (focal adhesion kinase), PAK (p21 activated kinase), and parvalbumin. Nucleic acid probe.
[6] The nucleic acid specified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193 nt to 936 nt of SEQ ID NO: 1 have been deleted. The nucleic acid probe according to any one of the above.
[7] (a) having a nucleotide sequence obtained by deleting a part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95 A nucleic acid or a nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above An expression vector for visualizing synaptic enhancement, comprising a multicloning site inserted between (b).
[8] The expression vector according to [7] above, wherein a nucleic acid encoding a reporter protein is inserted into the multicloning site.
[9] The nucleic acid encoding the reporter protein is selected from the group consisting of Venus, green fluorescent protein, channelrhodopsin, halorhodopsin, glutamate decarboxylase, luciferase, chloramphenicol acetyltransferase, β-galactosidase, and β-glucuronidase. The expression vector according to [8] above, which is a nucleic acid encoding one or more selected proteins.
[10] The expression vector according to [9], further comprising a nucleic acid encoding a protein selected from one or more of the group consisting of Rho family G protein, FAK, PAK, and parvalbumin.
[11] The nucleic acid identified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193nt to 936nt of SEQ ID NO: 1 have been deleted. The expression vector according to any one of the above.
[12] A method of visualizing synaptic enhancement by incorporating the expression vector according to any one of [8] to [11] into a nerve cell and expressing a reporter protein.
[13] A transgenic non-human animal in which the nucleic acid probe according to any one of [3] to [6] is introduced into a nerve cell.

  Since the probe of the present invention can specifically accumulate at an increased synapse, it is not necessary to observe the synaptic morphology, and it is possible to introduce a gene into all nerve cells and visualize the increase in synapse in a wide range and in a short time. it can.

FIG. 3 shows accumulation dependent on synaptic enhancement of probes of the invention in hippocampal slice cultures. Fig. 3 shows the accumulation characteristics of the probe of the present invention during motor learning in vivo. Figure 2 shows the spatio-temporal dynamics of the probe of the present invention during motor learning in vivo. FIG. 6 shows the reduction of spines containing a probe of the present invention by photoactivation. Deletion of acquired learning by photoactivation.

Hereinafter, preferred embodiments will be described in detail for the purpose of explaining the present invention.
The present invention encodes a nucleic acid having a base sequence encoding a PSD95 variant in which part or all of PSD-95, which is an ion channel receptor anchoring protein, is deleted, and a dendritic target element (DTE) A gene cassette containing a nucleic acid having a base sequence, a nucleic acid probe in which a nucleic acid encoding a reporter protein is inserted into the gene cassette, a protein probe in which a protein is expressed from the nucleic acid, an expression capable of expressing the probe in a nerve cell The present invention relates to a vector, a method for visualizing synaptic enhancement, and a transgenic non-human animal into which the nucleic acid probe has been introduced and which can monitor synaptic enhancement.

1. Object Visualized Using Probes of the Present Invention The present invention provides tools for visualizing synapses (especially spines) associated with memory and learning using nucleic acid probes or protein probes. Elucidation of the molecular mechanisms of synapse formation and plasticity not only reveals higher brain functions such as memory and learning, but also elucidates the pathophysiology of dementia, neuropsychiatric disorders, epilepsy, etc., and develops effective treatments It is thought to encourage. Synapses consist of two specialized structures, pre-synapse and post-synapse, and transmit signals in one direction. It is known that the accumulation of proteins unique to the presynapse and the postsynapse is essential for the formation and maintenance of this special structure. For example, SNAP-25 involved in neurotransmitter release is selectively placed at the post-synapse, while PSD-95 (Postsynthetic Density 95 kD), which is an anchoring protein of an ion channel receptor, is placed at the pre-synapse. Are known to be selectively placed. These SNAP-25 and PSD-95 undergo post-translational lipid modification called palmitoylation, and this lipid modification is known to control the specific localization and function of SNAP-25 and PSD-95. . In addition, transport of AMPA (α-amino-3-hydro-5-methyl-4-isoxazolepropionic acid) receptor, which is a receptor for glutamate, which is a major neurotransmitter of excitatory synapses in the brain, to the synapse. And recycling are controlled in a neural activity-dependent manner, suggesting that they are the main body of synaptic plasticity. Since PSD-95 anchors the AMPA receptor to the post-synapse via the main binding protein of AMPA receptor, stargazin, dynamic regulation of PSD-95 plays an important role in synaptic plasticity. It is said that there is. PSD-95 contains an N-terminal part, three PDZ domains, an SH (Src homology) domain, and a GK (guanylate kinase) domain in the molecule.

  When a specific post-synapse increases, the neural circuit that the increase group constitutes is used efficiently. As described above, the present inventors have developed PSD-95, which is a post-synaptic protein during synapse increase. Focusing on the increased synapse, and with the memory and learning, the mRNA of the Arc gene accumulates on the dendrites receiving neural inputs, and PSD-95 mutants (for example, PDZ domains 1 and 2). By using as a probe a protein fused with "PSD-95Δ1-2"), a fluorescent protein, and a dendritic target element (DTE) (part of the translation product from Arc mRNA). And the visualization of synaptic growth and neoplasia associated with memory. Therefore, this invention can visualize the synapse (especially spine) increased by receiving a certain stimulus or more. Below, probes and visualization methods for visualizing synaptic growth and neoplasia are detailed.

2. Gene Cassette The present invention provides a gene cassette for use in a nucleic acid probe (described later) that visualizes synaptic enhancement. According to the present invention, any gene cassette may be used as long as each nucleic acid is combined and encodes a plurality of proteins that correlate synaptic growth and neoplasia with brain function. Typically, the gene cassette of the present invention is not limited,
(A) a nucleic acid having a base sequence obtained by deleting part or all of 192 nt to 935 nt from the nucleic acid consisting of the base sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding the dendritic target element (DTE), or under a stringent condition with the nucleic acid. It is a gene cassette for visualizing synaptic enhancement, including a nucleic acid that hybridizes in

  The nucleic acids encoding PSD95 (and variants thereof) and DTE used in the gene cassette of the present invention include single-stranded or double-stranded DNA and RNA complements thereof. DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. As the nucleic acid used in the present invention, DNA is preferable. As is well known, codons are degenerate and some amino acids have a plurality of base sequences encoding one amino acid. However, any base sequence may be used as long as it is the base sequence of each nucleic acid encoding the protein. Nucleic acids having these are also within the scope of the present invention.

As used herein, “PSD-95” is a protein that reversibly binds to an ion channel type receptor, and is a MAGUK (membrane associated guanylate kinase) protein that exists after a synapse where spines are densely present. And has the function of structurally maintaining synapses. The nucleic acid encoding PSD-95 includes a base sequence of 2172 bases represented by SEQ ID NO: 1, and includes (i) N-terminal part (1 to 192 nt) and (ii) three PDK domains as characteristic domains. (PDZ1: 193-453 nt, PDZ2: 478-738 nt, PDZ3: 936-1179 nt), (iv) SH (Src homology) domain (1303-1485 nt), and (v) GK (guanylate kinase) domain (1600-2136 nt) ). The base sequence (1-272 nt) of PSD-95 is GenBank Accession No. NM This corresponds to 58 to 2229 nt in 019621.

  According to the present invention, when a nucleic acid encoding PSD-95 is used as part of the gene cassette, the full length of the nucleic acid encoding PSD-95 may be used, but it encodes DTE as part of the gene cassette. When using a nucleic acid, it is preferable that part or all of 193 nt to 936 nt in SEQ ID NO: 1 is deleted in the base sequence of PSD-95. As used herein, when a portion to be deleted is a part, this “part” means one of the above 193 to 936 nt, a base connected to or discontinuous 2 743 bases, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 , 100, 200, 300, 400, 500, 600, 700 bases. According to the present invention, a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193 to 936 nt (744 bases) have been deleted (in this case, the protein encoded by the nucleic acid is referred to as “PSD-95Δ1-2”. May be used). In one embodiment of the present invention, a nucleic acid encoding PSD-95 that can be used as a gene cassette includes a nucleic acid comprising a nucleotide sequence from which a part or all of 192 nt to 935 nt has been deleted in SEQ ID NO: 1 and a string Nucleic acids that hybridize under mild conditions are also included.

As used herein, a “dendritic target element (DTE)” is a protein that constitutes part of the Arc gene product that accumulates in dendrites that have received neural input with memory and learning. DTE is a protein that is translated from mRNA into protein in nerve cells, but is associated with synaptic plasticity, which is a basic neural function for forming learning and memory, and mRNA that encodes DTE directly under the plastic synapse Is known to be expressed and protein expressed. Therefore, in order to achieve the object of the present invention of visualizing synaptic enhancement, it is preferable as a nucleic acid used as a part of a gene cassette. The base sequence of DTE (SEQ ID NO: 3 to 644 nt) is GenBank Accession No. NM This corresponds to 2036 to 2699 nt in 019361. In one embodiment of the present invention, the nucleic acid encoding DTE that can be used as a gene cassette includes a nucleic acid that hybridizes with a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3 under stringent conditions. .

  For the nucleic acid encoding PSD95 (or a variant thereof) and DTE, for example, as described in Example 1 described later, synthetic nucleotides are designed as primers based on the nucleotide sequences of SEQ ID NOs: 1 (or 4) and 3, respectively. However, it can be prepared by amplification using a plasmid containing chromosomal DNA as a template. Examples of the method for amplifying a nucleic acid include polymerase chain reaction (PCR) (Saiki RK, et al., Science, 230, 1350-1354 (1985)), and ligase chain reaction (LCR) (Wu DY. Et al., Genomics, 4, 560-569 (1989)), and transcription-based amplification (Kwh DY et al., Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)). As well as strand displacement reactions (SDA) (Walker GT et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992); Walker GT et al., Nuc. Acids. Res., 20, 1691-1696 (1992)), self-retaining sequence replication (3SR) Guatelli J. C., Proc. Natl. Acad. Sci. USA, 87, 1874-1878 (1990)), and Qβ replicase system (Lizardi, PM et al., BioTechnology, 6, 1191-2202 (1988)). However, the present invention is not limited to these. In the present invention, it is preferable to use the PCR method. In addition, the method of introduce | transducing a variation | mutation into a gene is generally known, for example, a site-directed mutagenesis method (Current Protocols in Molecular Biology, edited by Ausubel, FM, et al., Unit 8.1-). 8.5, John Wiley & Sons, Inc., NY). In the present invention, QuikChange II XL Site-Directed Mutagenesis Kits (Agilent Technologies) can be used.

  As used herein, “under stringent conditions” means to hybridize under moderate or high stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of DNA. Basic conditions are described in Sambrook, J. et al. Et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001), with respect to nitrocellulose filters, 5 × SSC, 0.5% SDS, 1. Pre-wash solution of 0 mM EDTA (pH 8.0), about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or Stark solution (Stark in about 50% formamide at about 42 ° C.) use of other similar hybridization solutions) such as' s solution) and washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. Generally, such conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions, such as hybridization conditions as described above, and about 68 ° C. Defined with 0.2 × SSC, 0.1% SDS wash. One skilled in the art can recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as the length of the probe.

  Homologous nucleic acids cloned using the nucleic acid amplification reaction or hybridization as described above are at least 30% or more, preferably, at least 30% or more, respectively, with respect to the base sequences set forth in SEQ ID NOs: 1 (or 4) and 3 50% or more, more preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more. Note that the percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by Devereux et al., Nucl. Acids Res. , 12, 387 (1984) and determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3) available from the University of Wisconsin Genetics Computer Group (UWGCG). can do.

  As another embodiment of the present invention, the nucleic acid encoding PSD95Δ1-2 used in the present invention consists of a total length of 1428 bases as shown in the base sequence represented by SEQ ID NO: 4. As one embodiment of the present invention, a nucleic acid having an activity equivalent to that of PSD95Δ1-2 and having a base sequence in which 1 to 150 bases are substituted in the base sequence may be used. The number of substituted bases is preferably at most 100, more preferably at most 75, even more preferably at most 50, even more preferably at most 30, still more preferably at most 20, even more preferably at most 10 , Most preferably at most 5.

  As another embodiment of the present invention, the nucleic acid encoding DTE used in the present invention consists of a total length of 663 bases as shown in the base sequence represented by SEQ ID NO: 3. As one embodiment of the present invention, a nucleic acid having an activity equivalent to that of DTE and having a base sequence in which 1 to 70 bases are substituted in the nucleic acid sequence may be used. The number of substituted bases is preferably at most 60, more preferably at most 50, even more preferably at most 30, even more preferably at most 20, still more preferably at most 10, even more preferably at most 5 Most preferably, 1 is a maximum of 3.

  In the present invention, when a nucleic acid encoding PSD95 (and its variant) and DTE is used as a gene cassette, the order in which they are arranged is not limited, but the nucleic acid encoding PSD95 (and its variant) Preferably, it is arranged upstream of the nucleic acid encoding.

3. Nucleic Acid Probe According to the present invention, there is provided a nucleic acid probe in which a nucleic acid encoding a reporter protein is incorporated into the gene cassette in order to visualize synaptic enhancement. In one embodiment of the present invention, the insertion position of a nucleic acid encoding a reporter protein is not limited as long as it is incorporated in a nucleic acid probe and can function as a reporter protein. For example, a nucleic acid encoding a reporter protein may be inserted between a nucleic acid encoding PSD95Δ1-2 constituting a gene cassette and a nucleic acid encoding DTE. Thus, nucleic acid probes for visualizing synaptic enhancement of the present invention typically include
(A) a nucleic acid having a nucleotide sequence obtained by deleting part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above It is preferable that it contains the nucleic acid which codes the reporter protein inserted between (b). In addition, the definition of the nucleic acid in said (a) and (b) is equal to the definition of each corresponding nucleic acid used for a gene cassette.

  As used herein, “reporter protein” refers to a gene cassette contained in a nucleic acid probe, depending on the expression of brain function (learning or memory), when the nucleic acid probe of the present invention is introduced into a nerve cell. It means a protein that is translated together with the nucleic acid to be used to visualize the expression of brain function. Accordingly, the reporter protein is not limited as long as it is a protein that can be used for the above-mentioned purpose. For example, Venus, green fluorescent protein, channelrhodopsin, halorhodopsin, glutamate decarboxylase, luciferase, chloramphenicol acetyltransferase, β- Galactosidase and β-glucuronidase are included. In addition, the number and kind of the nucleic acid encoding the reporter protein inserted into the gene cassette are not limited, and two or more kinds may be inserted according to the purpose of use.

  In another embodiment, the present invention provides, as a nucleic acid to be introduced into a nucleic acid probe, in addition to the above-described nucleic acid encoding a reporter protein, various nucleic acids together with a nucleic acid encoding a reporter protein, depending on the research purpose. Nucleic acid probes introduced into can be provided. For example, a gene encoding a protein that is activated in response to light stimulation may be introduced. PaRac1 (Wu, YI, et al., Nature, 461, 104-108 (2009)), PaRhoA, RaCdc42, or the like may be used as a protein that is activated in response to such a stimulus. Rac1, RhoA, and Cdc42 are all Rho family G proteins, and are proteins that control the formation of a cytoskeleton (for example, actin, myosin, etc.) that controls cell morphology and movement. Here, for example, PaRac1 is a fusion protein in which a LOV domain (light, oxygen, voltage domain) whose structure is changed by light absorption in Avena sativa phototropin protein is fused to Rac1 protein (Wu et al., Supra). . When PaRac1 is expressed in neurons and exposed to blue light from the outside, local activation of Rac1 promotes actin polymerization through the activation of phosphorylase PAK, and the spatiotemporal spatial and temporal of cell morphology and movement Operations can be performed. Thus, since Rac1 can be activated by blue light, synaptic plasticity can be induced by artificial manipulation such as light stimulation. In another embodiment, the nucleic acid probe further comprises a nucleic acid encoding a protein selected from the group consisting of FAK (focal adhesion kinase), PAK (p21 activated kinase), and parvalbumin together with a nucleic acid encoding a reporter protein. Can be introduced. Here, FAK is a kind of integrin-binding protein, and is a kinase that is localized in a cell adhesion group and is involved in cell morphology and movement. PAK is a phosphorylating enzyme related to cytoskeleton construction. Parvalbumin is a calcium-binding low molecular weight albumin that is present in GABA-driven interneurons of the nervous system and has been found to reduce parvalbumin expression in patients with schizophrenia. .

4). Expression Vector According to the present invention, the gene cassette of the present invention is preferably used in the form of a vector incorporating the nucleic acid constituting them. More typically, the vector for visualizing synaptic enhancement of the present invention is:
(A) a nucleic acid having a nucleotide sequence obtained by deleting part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above It may be an expression vector containing a multiple cloning site inserted between (b). Here, the “multicloning site” means a site where sequences recognized by various restriction enzymes are present, which can be cleaved with an appropriate restriction enzyme and foreign nucleic acid can be inserted. A person skilled in the art can design a restriction site sequence that can be used as a multiple cloning site. According to the present invention, for the purpose of visualizing the increase in synapse, the foreign nucleic acid inserted into the multicloning site is not limited, but a nucleic acid encoding a reporter protein (described above) is preferable. In another embodiment, the present invention introduces various nucleic acids as a nucleic acid to be introduced into an expression vector, in addition to a nucleic acid encoding the reporter protein, together with a nucleic acid encoding the reporter protein, depending on the purpose of research. An expression vector can be provided. For example, it may be an expression vector into which a nucleic acid encoding a protein that is activated in response to light stimulation is introduced, and examples of such a protein include PaRac1 (see “3. Nucleic Acid Probe”). See). Furthermore, the expression vector may include, but is not limited to, a promoter (eg, CAG), an enhancer, a start codon, a stop codon, and a terminator in order to properly express a nucleic acid encoding the incorporated reporter protein. Good.

  In the present invention, when PSD95 (and its variant) and a nucleic acid encoding DTE are introduced into an expression vector as a gene cassette and used, there is no restriction on the order in which they are arranged, but PSD95 (and its variant) The encoding nucleic acid is preferably located upstream of the nucleic acid encoding DTE. Furthermore, the expression vector of the present invention preferably has a multicloning site between the nucleic acid encoding PSD95 (and its variants) and the nucleic acid encoding DTE. According to the present invention, a nucleic acid encoding a reporter protein (for example, Venus) and a nucleic acid encoding PaRac1 can be incorporated into this multicloning site by treating with a restriction enzyme. When a nucleic acid encoding a protein activated in response to light stimulation such as PaRac1 is incorporated into the expression vector of the present invention, due to the nature of the protein, a stop codon is provided upstream of the nucleic acid encoding DTE. It is preferable to incorporate the nucleic acid of the protein into.

  Examples of a method for incorporating a target nucleic acid into a vector include Sambrook, J. et al. Et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 1.1 (2001). For convenience, a commercially available ligation kit (for example, manufactured by Toyobo Co., Ltd.) can also be used. A vector can be prepared simply by ligating a desired gene to a recombination vector (for example, plasmid DNA etc.) available in the art by a conventional method. Although it does not limit as a vector used in order to introduce | transduce the nucleic acid probe for visualizing the increase in synapses of this invention, A viral vector is preferable. Usable viral vectors include adeno-associated viruses (AAV) (eg, AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7, AAV2 / 8 serotype vectors); vectors derived from adenovirus (AV) Retroviruses (eg, lentivirus (LV), rhabdovirus, murine leukemia virus); herpes virus; vesicular stomatitis virus (VSV) and the like.

  A person skilled in the art can appropriately select the restriction ends so as to be compatible with the recombinant vector, and can further appropriately select a recombinant vector suitable for the host cell in order to express the desired protein. . Such a vector is a region in which the gene used in the present invention functions so that homologous recombination occurs with the gene of the target host cell (if necessary, an autonomous replication origin, a conjugative transfer region, a selectable marker (for example, , Kanamycin resistance gene), etc.) are appropriately arranged or introduced so that the nucleic acid is constructed or constructed so that it is appropriately recombined.

  In general, a transformant can be produced by incorporating a recombinant vector into a host cell. In this case, the host cell can be a prokaryotic cell (eg, E. coli (S17-1 strain, etc.), Bacillus subtilis) or a eukaryotic cell (mammalian cell, yeast, insect cell, etc.). Introduction (transformation) of a recombinant vector into a host cell can be performed using a known method. For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)), the protoplast method (Mol. Gen. Genet., 168, 111 (1979)), competent method (J. Mol. Biol., 56, 209 (1971)), calcium chloride method, electroporation method and the like.

  According to the present invention, the expression vector prepared above may be directly introduced into the brain at a predetermined site (eg, cortex). A vector is introduced by such means, and it is useful for visualizing in real time an increase in synapse associated with a brain function of a living body by a measurement method described later. A method for introducing the expression vector into the brain is not limited, but a physiological saline containing the expression vector may be directly injected using a pipette.

5. Method and Detection for Visualizing Synaptic Enhancement According to the present invention, synaptic enhancement can be visualized using an expression vector incorporating the nucleic acid probe of the present invention. When the reporter protein is a fluorescent protein in order to observe a nerve cell in real time, without limitation, a two-photon laser microscope, a fluorescence microscope, or a confocal laser microscope can be used. Here, the “two-photon laser microscope” is a microscope that uses a two-photon absorption process for material excitation, and is excited by a long-wavelength laser beam that is at least twice the wavelength required for normal one-photon excitation. Since the scattering of light is inversely proportional to the fourth power of the wavelength, for example, with respect to 500 nm of the one-photon excitation laser, the scattering of the tissue is reduced to 1/16 times in principle with a 1000 nm laser beam used for two-photon excitation. A microscope with the advantage that the depth of the deep part that is at most several tens of μm from the tissue surface in normal fluorescent microscopes can acquire images of deep parts up to about 1 mm, and the phototoxicity is greatly reduced because of the long wavelength. It is. Due to the overwhelmingly advantageous characteristics for such living body observation, live imaging can be performed with a relatively low degree of invasiveness to the inside of a living brain. Furthermore, by analyzing the behavior of the same individual at the same time, it is possible to observe the phenomenon observed inside the brain and the phenomenon at the behavior level at the same time and explore the relationship between them.

6). Transgenic non-human animal According to the present invention, it is possible to provide a transgenic non-human animal used for visualizing and observing synaptic enhancement in real time. The transgenic non-human animal of the present invention is not limited as long as it achieves the above object, and its production method and other characteristics are not limited. More typically, it is a non-human transgenic animal transformed or homologously recombined into a chromosome using the expression vector of the present invention. Animals other than humans (non-human animals) are, for example, vertebrates, preferably mammals. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs and cats. Preferred are mouse and rat. Further, the transgenic non-human animal of the present invention should be understood by a wide concept including not only non-human animals but also a part of non-human animals, for example, cells, tissues, organs, etc. of non-human animals. is there.

  Using the expression vector of the present invention, the nucleic acid probe of the present invention is introduced into animal germ cells other than humans by the transgene method to produce transgenic animal cells. Next, transgenic animals other than humans can be produced by generating this. The “transgene method” is a method in which a plurality of copies of a transgene are inserted at unspecified positions in genomic DNA of a host. According to the present invention, for example, a transgene method using a CAG promoter + a nucleic acid encoding PSD-95Δ1-2 + a nucleic acid encoding Venus (a nucleic acid encoding PaRacl) + a nucleic acid encoding DTE is used as a transgene. preferable.

  In the present invention, the cell used as a host is not particularly limited as long as it is a non-human cell. For example, a non-human fertilized egg can be used. Hereinafter, a method for introducing a transgene into a non-human fertilized egg will be described in more detail. Non-human fertilized eggs used in the present invention are developed and grown by introducing a transgene into them, and a nucleic acid encoding PSD-95Δ1-2 + a nucleic acid encoding Venus (+ PaRac1 is encoded by the activity of the promoter region. Any nucleic acid can be used as long as it can produce a non-human transgenic animal that expresses a nucleic acid encoding + DTE, and is not particularly limited. Such a fertilized egg is obtained by mating males and females of animals other than humans. The obtained fertilized egg can be obtained by injecting a transgene by a microinjection method or the like, and artificially transplanting the animal into a female oviduct, implanting it, and giving birth. As the fertilized egg of the mouse, for example, those obtained by mating of mice derived from B6C3F1, C57BL / 6, 129 / sv, BALB / c, C3H, SJL / Wt and the like can be used.

  The amount of transgene is suitably 100 to 3,000 copies. Examples of methods for introducing a transgene include commonly used methods such as a microinjection method and an electroporation method. Here, selection of the pup mouse into which the transgene has been introduced is carried out by cutting off the tip of the mouse tail and extracting the polymer DNA (Genetic Engineering Experiment Manual, supervised by Tatsuji Nomura, edited by Motoya Katsuki, Kodansha (1987) ) Or DNAeasy Tissue Kit (manufactured by QIAGEN), etc., to extract genomic DNA and confirm the presence of the transgene in a commonly used method such as Southern blotting or PCR. it can. Furthermore, it can be confirmed by a commonly used method such as Northern blotting or Western blotting that the transgene actually introduced in the individual is expressed and a protein translated from the gene is produced. it can. In the present invention, when a gene encoding a fluorescent reporter protein is introduced, in the above example, the gene is introduced so as to express a fusion protein of PSD-95Δ1-2 + venus (+ PaRac1) + DTE. By this, the expression of PSD-95Δ1-2 and DTE can be visualized in the living body. The method for observing the visualization may be as described above, and preferably a two-photon laser microscope can be used.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples. Note that the experimental protocol using mice in this example was approved by the Ethics Committee on Animal Experiments at the University of Tokyo, and was published by the National Institutes of Health (NIH). Was implemented in accordance with the “Guidelines on Revision” (revised 1996).

Example 1: Construction of expression vectors Deletion of PSD-95 and PaRac1 is described in a previous report (Hayashi-Takagi, A., et al., Nature Neurosci., 13, 327-332 (2010)). Followed the method. In summary, PSD-95Δ1-2 is NM Referring to 019621, it was prepared by deleting 192 nt to 935 nt from SEQ ID NO: 1. DTE has the following primers (sequences 1 and 2):
SEQ 1: 5'-atgat aagctt tcggctccatgactcagccatgcc- 3 '( SEQ ID NO: 5) (underlined is the HindIII restriction site); and sequence: 5'-atgat aagctt agacacgagcagttaccaacacg-3 ' ( SEQ ID NO: 6) (The underlined portion is HindIII Restriction site)
Was used to clone from cDNA generated from the frontal cortex of SD rats. Next, NM An amplicon having a base sequence (SEQ ID NO: 3) corresponding to 2036nt to 2699nt of the sequence shown in 019361 was subcloned downstream immediately after the stop codon of PaRac1. The PSD-95Δ1-2 and DTE were inserted into restriction sites (NheI and HindIII) of the pCAG vector having a CAG promoter.

Example 2: Visualization of synaptic enhancement Method (1) Production of AAV2 / 5 Vector AAV virus was produced based on the AAV helper free system (Agilent technologies, CA). Each pAAV vector, pRep-Cap (AAV5; Applied Viromics, Calif.), And pHelper plasmid were cotransfected into AAV-293 cells by polyethyleneimine “Max” (Polysciences, Pa.). After 72 hours incubation, cells were harvested and lysed using 5 freeze-thaw cycles. The obtained supernatant was layered on a 40% sucrose solution containing 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.01% BSA (v / v), and then at 4 ° C. for 16 hours. Centrifugation at 100,000 g. The pellet (crude virus particles) was treated with 1000 U of Benzonase nuclease (Novagen, WI) at 37 ° C. for 1 hour. After removal of debris by filtration through a 5 μm filter, the filtered material was subjected to a CsCl gradient (1.25 g and 1.50 g / ml) at 257,300 g at 15 ° C. for 48 hours. The virus rich fraction was recovered and then the solvent was replaced with ASCF (1 mM MgCl ₂ , 10 mM HEPES, free of CaCl ₂ ). Viral titers were determined by quantitative real-time PCR analysis (SYBR Green; Takara Bio Inc, Japan).

(2) Virus injection and cranial open cranial window surgery The AAV2 / 5 vector prepared above was injected into the mouse cortex as follows. Adult C57BL / 6 mice were anesthetized with isoflurane with intraperitoneal injection of mannitol (4 μg / g body weight) and dexamethasone to prevent brain swelling. Ketoprofen (40 μg / g body weight) and penicillin / streptomycin (4 U / g body weight) were injected subcutaneously for 4 consecutive days before surgery to prevent inflammation. The skull was exposed throughout the area corresponding to the M1 cortex according to stereotaxic coordinates. 1 μl of AAV preparation (0.5-4.0 × 10 ¹² genome copies / ml) was added to a glass pipette (tip diameter) at a flow rate of 150 nl / min using a syringe pump (Legato 130; Muromachi Kikai, Japan). 30 μm, inclined at an angle of 45 °). To make the open skull window, stainless trephine (φ1.8 mm, Fine Science Tools, CA) was used to make a circular open skull window. In order to avoid brain damage, intermittent excavations were performed at 10,000 revolutions / minute, during which continuous perfusion was performed gently with oxygenated ACSF. Excavation-induced repression on the skull was maximally avoided. After confirming that no bleeding was observed, the drill hole was covered with a circular cover slip (φ1.8 mm, thickness less than 0.1 mm) (Matsunami Glass, Japan) and sealed with dental cement (Fuji Lute BC; GC, Japan) did. Next, headgear was mounted for in vivo imaging.

(3) Two-photon imaging, glutamate uncaging, and photoactivation Two-photon imaging was performed using an FV1000 laser scanning microscope system (FV1000, with a water immersion objective lens (LUMPlanFL N, x60, 1.0 NA)). An upright microscope (BX61WI; Olympus, Japan) equipped with Olympus was used. A bimode-locked femtosecond pulsed Ti: sapphire laser (MaiTai DeepSee and HP; Spectra Physics, CA) was used at 1000 nm for dual color imaging (Venus and mRFP) and 720 nm for glutamate uncaging. For three-color imaging for mTurquise / SEP-GluA1 / mRFP, two independently taken images at 780 nm (with mTurquise and mMRFP) and 970 nm (SEP-GluA1 and mRFP) are shown in common by mRFP. Overlay based on fluorescence signal. For in vitro imaging, 10-40 xy images (digital zoom × 5, 512 × 512 pixels) were captured with a 0.5 μm step size on the z-axis. For in vivo imaging, mice were anesthetized with isoflurane and images (digital zoom × 2, 1024 × 1024 pixels) were captured from the dura mater with a step size of 1.0 μm for 300 μm. For glutamate uncaging, 8 mm MNI-glutamic acid (Tocris, UK) in Mg ²⁺ -free ACSF containing 1 μM tetrodotoxin (TTX; Wako Pure Chemical Industries, Japan) was added to 10 μM forskolin (Wako) and 5 μM forskolin (Wako) ) And topically applied from a glass pipette onto the dendrites. Repeated (5 Hz, x80) photolysis of MNI-glutamic acid on the spine head was performed at 720 nm and the pulse time was 0.6 ms. The uncaging laser intensity was 6 mW under the objective lens. Two-photon photoactivation of PcGFP (which is photoconvertible) at 720 nm with 10 mW laser power was induced for circulating photoactivation for 10 ms including the entire spine head.

(4) In vivo photoactivation in freely moving animals Mice transduced by AAV injection or in utero gene transfer were subjected to normal open cranial window surgery to establish a bilateral glass window. It was done. An outer cylinder with an 18G needle without inclination (length 15 mm, inner diameter = 0.9 mm) was implanted in a glass window for later photoactivation. Prior to photoactivation, an optical fiber was inserted into the outer cylinder so that the fiber tip was placed directly on the cover glass and the connection between the fiber and the outer cylinder was securely locked with Blu-Tack. Nevertheless, it was easily desorbed after photoactivation. Light stimulation was performed using the COME-2 series (Lucir, Japan). It consists of a 457 nm laser diode, Optical swivels, and bilateral optics (COME2-α DF1; core diameter = 500 μm, 0.5 NA). The laser diode was adjusted to have an output of 20 mW at the end of each fiber. Light pulses were delivered for 1 hour at 1 Hz for 150 milliseconds, which was regulated by a customized LabView program (National Instruments, TX).

(5) Behavioral analysis Mice were housed under standard laboratory conditions (free access to food and water in a 12 hour light / dark cycle). All behavioral analyzes were conducted during the light period. As motor learning, RotaRod training (Rota-Rod Trademills ENV-576; Med associates, VT) was used. Prior to the training session, the mice were allowed to acclimatize to a stationary rod for 2 minutes. During the training period, a fixed speed protocol was applied at low speed (8 rpm) so that the mice hardly fell off the rod. After the mouse was reliably maintained on the rod, the speed was increased in steps until finally 40 rpm. Air was blown on the hind limbs as an aversive stimulus so that the mice learned easier and faster. As a result, the posture of the mouse was improved so that it was tilted forward rather than backward on the rod, and Massus easily performed this task at a higher speed. When falling, the mouse was immediately returned to the rod. Time to drop was automatically recorded. Three training sessions were conducted for 1-2 days (2 hours per session, for a total of 6 hours training). For evaluation of learning achievement, the rotarod “test” of the acceleration protocol (4 to 40 rpm) was tried three times at intervals of 5 minutes. The average of 3 trials was used as task performance. Subjects whose degree of improvement was less than 20% compared to the previous training performance were considered failures and were excluded from further analysis. The running speed of the mice was measured by a video tracking system (Lifelight 3; Actimetrics, IL).

2. Results (1) Visualization of synaptic enhancement in vitro Using the expression vector (nucleic acid probe) prepared in Example 1, visualization of synaptic enhancement in a hippocampal slice culture was attempted. Hippocampal slices (350 μm thick) were excised from SD rats using a vibratome (VT1200S; Leica, Germany). This was mounted on a 0.4 μm Millicell ™ culture insert (EMD Millipore, MA). 1.6 μm gold microcarriers were transfected into the hippocampal slices using a PDS1000 / He Biolistic gene gun (Bio-Rad, CA). Two to four days after transfection, hippocampal slice cultures were transferred to a recording chamber, using oxygenated artificial cerebrospinal fluid (ACSF, 95% O ₂ and 5% CO ₂ ) at 29-30 ° C. Constant perfusion was performed. Artificial cerebrospinal fluid comprises 125mM NaCl, 2.5mM KCl, 2mM CaCl 2, 1mM MgCl 2, 1.25mM NaH 2 PO 4, 26mM NaHCO 3, 20mM glucose, and 200 [mu] M Trolox the (Sigma-Aldrich, MO).

  The results are shown in FIG. FIG. 1a shows that a nucleic acid probe (C and E) containing PSD-95Δ1-2 (designated “PSDΔ1.2” in the figure) and a DTE sequence is necessary and sufficient for an unbalanced distribution of probes (middle). Arrowhead). FIG. 1b shows that the nucleic acid probe of the present invention (indicated as “AS-PaRac1-mRFP” on the third stage) accumulates in a part of the spine to the same extent as “SEP-GluA1” (second stage) of the prior art. I understand that. Neurons co-transfected with mTaq (mTurquoise), SEP-GluA1, and AS-PaRac1-mRFP were allowed to express genes for 36 hours. During 36 hours, enhanced spine was visualized by SEP-GluA1 (arrowhead). FIG. 1c images a single spine enhancement by glutamate uncaging with and without forskolin (FSK) and anisomycin. Although spine was increased by glutamate uncaging in the presence of forskolin (middle of FIG. 1c), this disappeared in the presence of anisomycin, so that the nucleic acid probe was able to synapse the protein expression (accumulation) from the probe. Correlating with the increase, it was suggested that the increased synapse can be specifically visualized.

(2) Visualization of synaptic enhancement in vivo The accumulation of nucleic acid probes in spines based on motor learning of mice was examined. Gene expression from the nucleic acid probe was transcribed under the control of the synaptic activity response element (SARE) and the Arc promoter. The Arc promoter is a promoter that promotes activity-dependent transcription in complex neurons in the cortical layers II / III of the primary motor area (M1). Neuronal long-term potentiation is induced by motor learning. The mouse cortex was injected with an expression vector containing the nucleic acid probe, and the activation of neurons induced by learning using rotarod was quantitatively compared before and after learning. Detection of fluorescence from the reporter protein was performed using a two-photon laser microscope. The result is shown in Fig. 2. In the figure, “Enlarged” indicates an increase in spine, and means an increase in spine head volume of 50% or more. “New” means a spine newly appearing by learning. “Shrunk” indicates the reduction of the spine, and means that the spine head volume decrease is 50% or more. “Eliminate” means the disappearance of the spine. “No changed” means that the increase and decrease of the spine are maintained without being observed, and the increase and decrease of the spine head volume are less than 50%. As can be seen from this figure, the nucleic acid probe specifically labeled newly appeared spine and increased spine, and the respective sensitivities were 83 ± 7.9% and 69 ± 3.0%. This suggests that the nucleic acid probe of the present invention accumulates specifically at synapses enhanced by learning and can be visualized with very high sensitivity.

  Next, we examined the spatiotemporal dynamics of nucleic acid probe accumulation in response to learning in mice in vivo. Nucleic acid probes were loosely transduced into II / III complex neurons by intrauterine electroporation. A cranial window was made to cover the M1 cortex. One month after the creation of the cranial window, imaging was attempted before and after rotarod training. In the panel of FIG. 3, the “Before” learning column shows the enhanced spine identified by the nucleic acid probe that was present one day before learning, in turn immediately after learning (the “learning” period), Shows enhanced spine after 1 day (“After-1”) and 2 days after learning (“After-2”). Of particular note is that synaptic enhancement is prominent immediately after learning by rotarod training (day 0), and it can be seen that spine is increased after learning (after two days).

Subsequently, to demonstrate the need for synapses for learning, we utilized the decrease that long-term activation of Rac1 significantly induces spine contraction. FIG. 4 shows a schematic diagram (FIG. 4a) and a typical image (FIG. 4b) of the experimental means by photoactivation of the neocortex with the head fixed. Also, a schematic diagram (Fig. 4c) and a z-stack image (Fig. 4d) of experimental means for photoactivation of freely moving animals are shown. Nucleic acid probe incorporating PaRac1 was introduced into mouse phase II / III neurons by intrauterine electroporation to perform rotarod training. In each hemisphere, at least 0.2 mm ² area was irradiated with light (465 nm). As a result, it can be seen that the spine into which the nucleic acid probe is introduced is reduced by light irradiation. On the other hand, spines into which the probe was not introduced were not affected by light irradiation.

  Furthermore, we examined the correlation between the spine reduction increased by learning and the motor learning ability of mice when light was irradiated to mice after rotarod training. FIG. 5a shows the experimental design. After injecting the AAV vector into M1 of the mouse, rotarod training was performed. It was divided into three groups (protocols 1 to 3) according to different light irradiation periods. Regarding the improvement of task performance after rotarod training, the time until the mouse fell was measured before training, immediately after training, and after light irradiation. FIG. 5b is divided into three groups (left: mRFP alone (control), middle: nucleic acid vector without DTE, right: nucleic acid with DTE), depending on the vector introduced into the mouse performed in protocol 1. vector). FIGS. 5c and d correspond to Procols 2 and 3, respectively, but the introduced vectors were both nucleic acid vectors containing DTE. In FIG. 5c, the fall time of the mouse was remarkably improved after training as compared to before training, but the light irradiation one day after training shortened the fall time to the same level as before training. It turns out that learning is not well established in the day. On the other hand, in FIG. 5d, it can be seen that the learning has been fixed without shortening the fall time even when the light is irradiated two days after the training.

  All publications and patent documents cited herein are hereby incorporated by reference in their entirety. While specific embodiments of the invention have been described herein for purposes of illustration, it will be apparent to those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (13)

(A) a nucleic acid having a base sequence obtained by deleting part or all of 192 nt to 935 nt from the nucleic acid consisting of the base sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (b) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding the dendritic target element (DTE), or under a stringent condition with the nucleic acid. A gene cassette for visualizing synaptic enhancement, comprising a nucleic acid that hybridizes with.   The gene cassette according to claim 1, wherein the nucleic acid identified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193nt to 936nt of SEQ ID NO: 1 have been deleted. (A) a nucleic acid having a nucleotide sequence obtained by deleting part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above A nucleic acid probe for visualizing synaptic enhancement, comprising a nucleic acid encoding a reporter protein inserted between (b).   The nucleic acid encoding the reporter protein is at least one from the group consisting of Venus, green fluorescent protein, channelrhodopsin, halorhodopsin, glutamate decarboxylase, luciferase, chloramphenicol acetyltransferase, β-galactosidase, and β-glucuronidase The nucleic acid probe according to claim 3, which is a nucleic acid encoding a selected protein.   The nucleic acid probe according to claim 4, further comprising a nucleic acid encoding a protein selected from one or more of the group consisting of Rho family G protein, FAK (focal adhesion kinase), PAK (p21 activated kinase), and parvalbumin. .   The nucleic acid specified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193 nt to 936 nt of SEQ ID NO: 1 have been deleted. Nucleic acid probe. (A) a nucleic acid having a nucleotide sequence obtained by deleting part or all of 193 nt to 936 nt from the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 encoding the ion channel receptor anchoring protein PSD-95, or A nucleic acid that hybridizes with the nucleic acid under stringent conditions;
(B) a nucleic acid comprising the base sequence represented by SEQ ID NO: 3 encoding a dendritic target element (DTE), or a nucleic acid that hybridizes with the nucleic acid under stringent conditions; and (c) (a) above An expression vector for visualizing synaptic enhancement, comprising a multicloning site inserted between (b).   The expression vector according to claim 7, wherein a nucleic acid encoding a reporter protein is inserted into the multicloning site.   The nucleic acid encoding the reporter protein is at least one from the group consisting of Venus, green fluorescent protein, channelrhodopsin, halorhodopsin, glutamate decarboxylase, luciferase, chloramphenicol acetyltransferase, β-galactosidase, and β-glucuronidase The expression vector according to claim 7, which is a nucleic acid encoding the selected protein.   The expression vector according to claim 9, further comprising a nucleic acid encoding a protein selected from one or more of the group consisting of Rho family G protein, FAK, PAK, and parvalbumin.   The nucleic acid specified in (a) is a nucleic acid having a base sequence (SEQ ID NO: 4) from which all of 193 nt to 936 nt of SEQ ID NO: 1 have been deleted. Expression vector.   A method for visualizing synaptic enhancement by expression of a reporter protein by incorporating the expression vector according to any one of claims 8 to 11 into a nerve cell.   A transgenic non-human animal in which the nucleic acid probe according to any one of claims 3 to 6 is introduced into a nerve cell.