JP2013040894A - Method for evaluating whether non-hominid animal is in state of having normal learning abilities or not, and medicine screening method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating whether a non-hominid animal is in a state of having normal learning abilities or not by analyzing and interpreting the learning abilities and brain state of the animal on the basis of a concept different from conventional LTP.SOLUTION: A method for evaluating whether a non-hominid animal is in a state of having normal learning abilities or not includes a learning step of causing the animal to take learning action, a detection step of examining a localized state of CapZ in brain neurons of the animal, and an evaluation step of evaluating the brain state through observation of the localized state of CapZ.

Description

  The present invention relates to a method for evaluating whether or not a non-human animal has a normal learning ability. More specifically, the present invention relates to a method for evaluating whether or not the brain of the animal can be normally learned by examining the localization of CapZ in the nerve cells of the brain of the non-human animal.

  In the case of conventional drug screening, a test method using changes in rodent behavior as an index is generally used to determine memory ability and learning ability. For example, in the case of “context-dependent fear conditioning contextual learning”, an animal placed in a place where pain has been given in the past is used to calculate the time rate of the fear reaction that expresses the pain, and the “freezing behavior” that stops moving at all. Evaluate as learning ability.

  In addition, in order to study the fundamental mechanisms of memory and learning at the neuronal and molecular level, LTP (Long Term Potentiation) using section specimens in vitro is mainly used as research material. The increase in current due to AMPA-type glutamate receptors observed in LTP is considered to be an elementary process of memory (Non-Patent Documents 1 and 2), and applied research to screening for drugs that affect memory and learning. Is also thriving. It should be noted that a phenomenon that should appear as an elementary process of memory (a phenomenon that indicates plasticity of a neural circuit) may occur only infrequently. The inventors' past research (Non-Patent Documents 3 and 4) also suggests that the number of spines that change due to stimuli that form a memory may be about several percent of the total.

“Memory enhancement: the search for mechanism-based drugs.” Lynch G. Nat Neurosci. 2002 Nov; 5 Suppl: 1035-8. “Learning from LTP: a comment on recent attempts to identify cellular and molecular mechanisms of memory.” Learn Mem. 1996 Sep-Oct; 3 (2-3): 61-73. “Experience-dependent, rapid structural changes in hippocampal pyramidal cell spines.” Kitanishi T, Ikegaya Y, Matsuki N, Yamada MK. Cereb Cortex. 2009 Nov; 19 (11): 2572-8. Epub 2009 Feb 24. “Contextual learning induces an increase in the number of hippocampal CA1 neurons expressing high levels of BDNF.” Chen J, Kitanishi T, Ikeda T, Matsuki N, Yamada MK. Neurobiol Learn Mem. 2007 Nov; 88 (4): 409-15 Epub 2007 Aug 31.

  The test methods used to determine the learning ability of animals used in conventional drug screening are subject to disease model animals even if they are appropriate when healthy animals are used as test subjects. In some cases, it is difficult to discriminate and interpret whether only the behavior has changed or whether the learning ability has changed. For example, when studying depression model animals or schizophrenia model animals that are thought to have poor learning ability, is the conventional test method in a state of immobility due to dullness or lethargy? It is difficult to discriminate whether it is immobile due to fear reaction. Thus, especially in the case of disease model animals, immobility of a cause different from learning can be considered, so it is not certain that the conventional test method really reflects learning ability. In addition, when screening a drug, the administered test drug has side effects and learning has been achieved with the test drug, even though the drug has become immobile due to a malfunction. There is also a danger of being misinterpreted as being in a stationary state because of

  Therefore, as a method for avoiding the uncertainty of the conventional learning test, a method of directly analyzing the change occurring in the memory charge portion of the brain is desirable.

  In order to capture the changes that occur in the memory area of the brain, methods using LTP have been generally considered. However, recent progress in research has raised doubts about the study of elementary processes of memory based on LTP formation. In the first place, stimuli that have been used in conventional LTP research to directly ignite neurons directly in vitro are not the same as stimuli that are naturally input to neurons during animal activity and learning. It is becoming clear that the artificial stimulus is not likely to reproduce the natural stimulus in the living body. Also, due to technological progress in recent years, the method of observing LTP has evolved dramatically, and if LTP formation occurs in vivo, the observation results should be reported reliably. Nevertheless, no report has been made that a phenomenon similar to LTP has occurred in vivo.

Based on these facts, while having a molecular mechanism partially in common with LTP that has been studied so far, new phenomena that occur due to natural stimuli occurring in the living body are returned to the origin of correlation with memory and confirmed. There is a need.
Traditional experimental approaches that have attempted to understand the mechanism by which memory is formed have been either molecular science, which looks at the entire brain, or physiology, which looks at some randomly selected neurons. When research is conducted using only such experimental methods, there is a risk of overlooking changes (phenomena) that occur in a small number of specific neurons, which may be important in the mechanism by which memory is formed.

  The present invention has been made in view of the above circumstances, and is based on a concept different from conventional LTP. By analyzing and interpreting the learning ability of an animal and its brain state, a non-human animal is normal. It is an object of the present invention to provide a method for evaluating whether or not a student has a sufficient learning ability.

  As a result of intensive studies by the present inventors, it has been found that there is a correlation between the formation of memory associated with learning by an animal and the localization of CapZ in nerve cells of the brain of the animal. Based on this finding, the following method is provided.

The method for evaluating the learning ability of a non-human animal according to claim 1 of the present invention is a method for evaluating whether or not a non-human animal is in a state having normal learning ability. A learning step for performing an action, a detection step for examining a localized state of CapZ in nerve cells of the brain of the animal, and an evaluating step for evaluating the brain state based on observation of the localized state of CapZ It is characterized by including.
The evaluation method according to claim 2 of the present invention is the evaluation method according to claim 1, wherein, in the evaluation step, when CapZ is localized in a part of the spine, the animal has a normal learning ability. It is characterized by evaluating.
The evaluation method according to claim 3 of the present invention is the method according to claim 1 or 2, wherein the non-human animal is introduced with a gene encoding a fusion protein in which CapZ and a marker protein and / or a peptide tag are bound. Further, the present invention is a transgenic animal, characterized in that the localization of CapZ is examined by detecting a signal derived from the marker protein and / or a signal derived from an antibody that specifically binds to the peptide tag. .
The evaluation method according to claim 4 of the present invention is the method according to claim 3, wherein the expression of the fusion protein in the transgenic animal is induced by the input in a brain region that receives the input in accordance with the learning behavior. Features.
The evaluation method according to claim 5 of the present invention is characterized in that, in any one of claims 1 to 4, the animal is a disease model animal.
The evaluation method according to claim 6 of the present invention is characterized in that, in any one of claims 1 to 5, CapZ is the following protein (i) or (ii).
(i) A protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5.
(ii) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and spine depending on input to a neuron; A protein that has a function localized in.
The method for screening a medicine according to claim 7 of the present invention comprises a step of administering a test substance to the disease model animal according to claim 5 exhibiting a learning disorder or a mental disease, and a learning behavior for the animal. A learning step for performing, a detection step for examining the localization of CapZ in nerve cells of the brain of the animal, and an evaluation step for evaluating the state of the brain based on the localization of CapZ;
In the evaluation step, when CapZ is localized in some spines, it is determined that the test substance is useful as a medicament for treating or preventing the learning disorder or psychiatric disorder And

According to the evaluation method of the present invention, it is possible to evaluate whether or not the brain of an animal that has performed a learning action has a normal learning ability that can accumulate (memorize) the memory related to the learning. By using the evaluation method of the present invention, it is possible to provide a new research base based on a phenomenon different from LTP formation regarding learning and memory of animals.
According to the pharmaceutical screening method of the present invention, a substance that is a drug candidate useful as a medicine for treating or preventing a learning disorder or a mental illness can be found.

2 is a schematic diagram of genes introduced into the transgenic mice used in Example 1. FIG. It is a photograph which shows the green fluorescence signal derived from EGFP-CapZ detected by observing the brain section | slice of the transgenic mouse used in Example 1 with the fluorescence microscope. FIG. 3 is a photograph showing a spine stained with red fluorescence of Rhodamine-phalloidin in the same visual field as in FIG. 2. The left is a photograph showing a fluorescence signal derived from the same EGFP-CapZ as in FIG. 2, and the right is a photograph showing the fluorescence signal observed by Rhodamine-phalloidin staining as in FIG. FIG. 3 is a photograph showing a dendrite in which MAP2 exists in the same observation visual field as FIG. 2 superimposed on the signal of EGFP-CapZ in FIG. 2. In the same observation visual field as FIG. 5, it is the photograph which showed only the dendrite in which MAP2 exists except the signal derived from EGFP-CapZ. FIG. 3 is a photograph observed with a wider field of view than FIG. 2 (observed without zooming). It is a photograph which shows the green fluorescence signal derived from EGFP-CapZ detected by observing the brain section | slice of the transgenic mouse used in the comparative example 1 with the fluorescence microscope. FIG. 9 is a photograph in which the signal intensity of the entire image is lowered in the same photograph as FIG. 8. The left is a photograph of phalloidin staining in the same visual field as in FIG. 7, and the right is a photograph of phalloidin staining in the same visual field as in FIG. 2 is a photograph showing a signal derived from an anti-CapZ antibody in a brain section of a wild-type mouse used in Example 2. 4 is a photograph showing a signal derived from an anti-CapZ antibody in a brain section of a wild-type mouse used in Comparative Example 2. 6 is a photograph showing a signal derived from an anti-CapZ antibody in a brain section of a disease model mouse used in Comparative Example 3. It is a graph showing the learning ability of the transgenic mouse which performed fear conditioning.

The present invention will be described below based on preferred embodiments with reference to the drawings.
<Evaluation method>
The method for evaluating the learning ability of a non-human animal of the present invention is a method for evaluating whether or not a non-human animal is in a state having normal learning ability, wherein the learning step causes the animal to perform learning behavior. And a detection step of examining the localized state of CapZ in the nerve cells of the animal brain, and an evaluation step of evaluating the brain state based on observation of the localized state of CapZ.
Steps other than the learning step, the detection step, and the evaluation step are incorporated as necessary.

In the present invention, the basis for evaluating and judging that "the animal is in a state having normal learning ability (was)" or "the animal is not in a state having normal learning ability (was not)"
“An animal that has previously confirmed that it has normal learning ability by another method accumulates memory by performing learning behavior in the learning process of the present invention” and “a signal is input along with the learning behavior. In other neurons, CapZ is localized in a part of spines in a neuron, and there is a high correlation between them and "an animal that has previously confirmed that it has a learning disorder and does not have normal learning ability". , “Do not accumulate memory by performing learning actions in the learning process of the present invention” and “when normal, CapZ is localized in some spines in the nerve cells to which signals are input along with the learning actions. `` Not present '' has a high correlation,
The present inventors have found out.

  In the present specification and claims, “CapZ is localized in some spines” means that the brain region where a signal (action potential) is input in accordance with learning behavior The concentration of CapZ in some spines contained in the spine is higher than the concentration of CapZ in the dendritic body having the spine, and there is a clear bias in the concentration of CapZ between the spines contained in the neural circuit. Means that. Usually, the concentration of CapZ is reflected with a positive correlation with the signal intensity derived from CapZ.

The non-human animal in the present invention is not particularly limited as long as it is an animal that allows learning behavior to be performed and accumulates memory relating to the learning behavior, for example, rodents such as mice, rats, guinea pigs, rabbits, Examples include primates such as monkeys and orangutans, mammals such as pigs, cows, horses, dogs, cats and dolphins, birds such as parakeets and juvenile pine, and fishes such as carp and zebrafish. Among these, primates, mammals, and rodents that can learn and memorize relatively high learning behavior are preferable, and rodents that are easy to breed and that are also used for screening drug candidate compounds are more preferable.
As the animal, an animal into which endogenous or exogenous CapZ has been introduced in advance can be used. The introduced animal will be described in detail later.
In addition, it is possible in principle to apply the evaluation method of the present invention to humans, but sufficient consideration should be given to safety and ethical issues.

[Learning process]
The learning step in the evaluation method of the present invention is a step of causing the animal to perform learning behavior.
The learning behavior is not particularly limited as long as the animal can memorize the stimulus by applying the stimulus to the animal. For example, when an animal is placed under certain conditions, it gives the animal a comfortable or unpleasant somatic sensation, thereby associating the situation where the animal is placed under the certain conditions with the somatic sensation. One example is “conditioning” that allows you to learn and understand that the two are related. As a conventional method using the “conditioning”, there is “context-dependent fear conditioning learning” that has been used in research related to memory and learning.

  As a typical implementation method of the conventional “context-dependent fear conditioning context learning”, conditioning is performed by combining an electrical shock (unconditional stimulus) and a neutral stimulus (conditional stimulus) such as a visual stimulus obtained from a place where an animal is present. This is a method for evaluating the fear reaction (shrinking behavior) expressed when the established animal is placed again in the place where the electric shock is given as context learning. Specifically, an animal is placed in a chamber in which an electric grid is installed, and electrical stimulation that feels pain is given for a short time. After a certain period of conditioning (1 hour, 24 hours, etc.), the freezing behavior in the conditioning chamber without electrical stimulation is measured for 2 to 5 minutes, and the percentage (%) of the freezing action time relative to the total measurement time is calculated. Is an index of learning ability

In the evaluation method of the present invention, when making an animal perform a learning behavior, whether or not the learning behavior is stored in the animal is different from the conventional method in that it is not evaluated / determined in the “learning step” stage. To do. In other words, in conventional context-dependent fear conditioning learning, after a specific condition (conditional stimulus) and electrical stimulation are presented in combination, the reaction of the animal when the specific condition is presented again after a predetermined time (shrinking behavior) Is analyzed. On the other hand, when context fear conditioning is performed in the learning process of the present invention, the method of presenting a combination of the specific condition and electrical stimulation is performed in the same manner, but the specific condition is presented again after a predetermined period of time and the animal's response There is no need to do the analysis.
That is, the evaluation method of the present invention does not evaluate / determine whether or not the learning has been established and the animal has accumulated memory regarding the learning behavior in the learning step. The state of the brain is directly observed, and in the evaluation step, it is evaluated / determined whether or not the animal has accumulated memory regarding its learning behavior.
Therefore, in the learning process of the present invention, the learning behavior to be performed by the animal is conventionally used, and the learning behavior in which it is confirmed in advance that the learning behavior can be stored by the animal is preferable. Such learning behaviors include, for example, Y-shaped maze test, rotarod test, fear conditioning learning test, water search test, novel substance search test, passive avoidance test, radial maze test, Morris water maze test and delayed sample matching. -The method of the part concerning presentation of a specific condition and a specific stimulus in conventional methods, such as a non-sample comparison test, can be illustrated as a suitable thing.

[Detection process]
The detection step in the evaluation method of the present invention is a step of examining the localized state of CapZ in the nerve cells of the animal brain.
As a method for examining the localized state of the CapZ, the brain of the animal that has undergone the learning process is directly observed after a predetermined time, or is chemically fixed and removed, and a section including a brain part related to the learning behavior A method of manufacturing Caps and examining the distribution of CapZ is suitable.

The predetermined time is not particularly limited, and may be a time at which memory related to learning behavior is considered to be accumulated in the brain region.
When the learning behavior is fear conditioning and the hippocampus of the animal is analyzed, the predetermined time is preferably 4 hours to 72 hours, more preferably 8 hours to 48 hours, and even more preferably 12 hours to 24 hours.
By being in the above range, it is considered that memory regarding learning behavior is accumulated in the hippocampus of the animal, and the localization of CapZ can be detected as a phenomenon highly correlated with the memory. If the brain is examined for a time less than the lower limit of the above range, it may be difficult to interpret the signal in the subsequent evaluation step. The memory is thought to be retained in the hippocampus for several days to several weeks, but when it is examined at a time exceeding the upper limit of the above range, another new memory is overwritten, thereby localizing the learning-dependent CapZ. May not be able to detect the signal indicating.

Known methods can be applied to the method of directly observing the brain, the method of chemically treating and removing the brain, and the method of preparing a brain slice.
There are no particular restrictions on which site to examine among the many sites in the brain. It is preferable to examine the state of the brain region highly relevant to the learning behavior to be performed by the animal in the learning process. For example, it has been reported that learning memory related to spatial cognition is accumulated in the hippocampus in the brain. Therefore, by examining the hippocampus in the brain of an animal that has made a learning action related to spatial cognition, it is possible to capture a phenomenon that correlates with the accumulation of memory related to the learning action.
In the evaluation method of the present invention, the phenomenon that correlates with the accumulation of memory is a phenomenon in which a protein called CapZ is localized in a part of the spine constituting the brain region or nerve cell.

  CapZ is a protein of about 270 to 290 amino acid residues, also called F-actin capping protein, and was originally identified as a protein having a function of binding to F-actin. Subsequently, the present inventors examined the possibility that CapZ is involved in synaptic plasticity of neurons. As a result, the present inventor confirmed that an event in which an animal accumulates memory by performing a learning action and an event in which at least a part of CapZ is localized in a part of a spine in a neuron that fires with the learning action. And have a high correlation.

  Depending on the organ of the animal, different isotypes of CapZ may exist, and these isotypes function by forming a homocomplex or heterocomplex. For example, at least three isotypes of CapZ beta1, CapZ beta2, and CapZ alpha have been confirmed in mice. Among these, CapZ beta2 and CapZ alpha are mainly expressed in the mouse brain.

CapZ in the present invention may be endogenous CapZ of the animal, or exogenous CapZ introduced into the animal by gene introduction or protein introduction.
The exogenous CapZ is not particularly limited as long as it has a function of localizing in spines and / or a function of binding to F-actin in nerve cells depending on signal input, and is derived from the same species of animal. CapZ may be sufficient and CapZ derived from a different animal may be sufficient. Further, it may be a mutant CapZ in which one or several amino acid residues are deleted, substituted, or added in the amino acid sequence of CapZ derived from the same or different animal species. Furthermore, a fusion protein in which a marker protein and / or a peptide tag is bound to CapZ may be introduced as exogenous CapZ.
The exogenous CapZ is preferably expressed in the brain of the animal from which it is derived. In addition, when CapZ derived from a different animal is introduced, the different animal is preferably closely related to the animal to be introduced.

  The cDNA information of CapZ cloned from animals can be viewed in a batch at NCBI's data bank UniGene. The ID number of CapZ alpha is UGID: 905681, and the ID number of CapZ beta2 is UGID: 256943. CapZ that can be searched in this data bank and linked data bank can be introduced as the exogenous CapZ.

  The homology of the amino acid sequence of CapZ is very high between animals belonging to mammals. For example, the homology of amino acid sequences compared between human and mouse is 98% for subunit alpha and 100% for subunit beta. For this reason, in mammals, it is considered that the introduced CapZ functions in an animal into which CapZ derived from a different animal is introduced.

When using CapZ derived from a mouse (Mus musculus) as the exogenous or endogenous CapZ, the CapZ is:
A protein of 272 amino acid residues (CapZ beta 2) represented by SEQ ID NO: 1 in the sequence listing;
A protein of 286 amino acid residues (CapZ alpha) represented by SEQ ID NO: 2 in the Sequence Listing;
A function of having one or several amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and localized in a spine in an input-dependent manner to nerve cells; A protein having a function of binding to F-actin;
A protein having a homology with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of 90% or more and having a function of localizing in spines and / or a function of binding to F-actin depending on input to nerve cells is preferable. .

  The amino acid sequence of SEQ ID NO: 1 (CapZ beta2) is the same sequence as Mus musculus capping protein (actin filament) muscle Z-line, beta (Capzb), transcript variant 2, mRNA registered in GenBank Accession No. NM_009798 is there. The amino acid sequence of SEQ ID NO: 2 (CapZ alpha) is the same sequence as Mus musculus capping protein (actin filament) muscle Z-line, alpha 1 (Capza1), mRNA registered in GenBank Accession No. NM_009797.2 .

When using CapZ derived from rat (Rattus norvegicus) as the exogenous or endogenous CapZ, the CapZ is:
A protein of 272 amino acid residues represented by SEQ ID NO: 3 in the sequence listing (CapZ beta);
In the amino acid sequence of SEQ ID NO: 3, one or several amino acids have an amino acid deleted, substituted or added, and function to localize spine depending on input to nerve cells and / or F-actin A protein having the function of binding;
A protein having a homology with the amino acid sequence of SEQ ID NO: 3 of 90% or more and a function of localizing in spines and / or a function of binding to F-actin in an input dependent manner to nerve cells is preferable.

  The amino acid sequence of SEQ ID NO: 3 (CapZ beta) is the same sequence as Rattus norvegicus capping protein (actin filament) muscle Z-line, beta (Capzb), mRNA registered in GenBank Accession No. NM_001005903.1.

When human-derived CapZ is used as the exogenous or endogenous CapZ, the CapZ is a protein of 272 amino acid residues (CapZ beta1) represented by SEQ ID NO: 4 in the Sequence Listing;
A protein of 286 amino acid residues represented by SEQ ID NO: 5 in the sequence listing (CapZ alpha1);
A function of having one or several amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 and being localized in a spine in an input-dependent manner to nerve cells; and / or A protein having a function of binding to F-actin;
A protein having a homology with the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 of 90% or more and having a function of localizing in spines and / or a function of binding to F-actin depending on input to nerve cells is preferable. .

  The amino acid sequence of SEQ ID NO: 4 (CapZ beta1) is identical to Homo sapiens capping protein (actin filament) muscle Z-line, beta (CAPZB), transcript variant 1, mRNA registered in GenBank Accession No. NM_004930.3 Is an array. The amino acid sequence of SEQ ID NO: 5 (CapZ alpha1) is the same sequence as homo sapiens capping protein (actin filament) muscle Z-line, alpha 1 (CAPZA1), mRNA registered in GenBank Accession No. NM_006135.2 .

  In the prepared brain slice specimen, a method for examining in which region CapZ is localized is not particularly limited, and a conventionally known method can be applied. For example, an immunostaining method using an antibody that selectively (specifically) binds to CapZ can be applied. A commercially available antibody may be purchased and used, or an anti-CapZ antibody may be prepared and used according to a conventional method.

  In order to examine in which part of the nerve cell CapZ is distributed, it is preferable to stain the structure constituting the nerve cell. For example, when the section is stained using an anti-MAP2 antibody, the position, shape and contour of dendrites where MAP2 is present can be observed. Further, when the section is stained using Rhodamine-phalloidine, the position, shape and contour of the spine containing F-actin can be observed. Therefore, by confirming the position and size of dendrites and spines in the observation field of the section, and detecting a signal derived from CapZ (fusion protein) in the same field, CapZ is located in any part of the nerve cell. It can be examined whether it is distributed or where CapZ is localized in the nerve cell.

As another more preferable method, the use of a transgenic animal into which CapZ is introduced (hereinafter sometimes referred to as CapZ-TG animal) can be exemplified.
The CapZ-TG animal is preferably a gene into which a gene encoding a fusion protein in which CapZ is bound to a marker protein and / or a peptide tag is introduced.
Since the marker protein or peptide tag is bound to the fusion protein, it is preferable to use the fusion protein because detection becomes easier. The CapZ-TG animal can be prepared by a conventional method.

  A method for detecting a primary antibody selectively bound to CapZ and a method for detecting the marker protein or peptide tag in a slice of an animal brain that has undergone a learning process and a method for detecting the marker protein or peptide tag are not particularly limited, and known methods can be applied. Examples thereof include a method of detecting with a fluorescence microscope using a fluorescently labeled secondary antibody that binds to the primary antibody, and a method of detecting fluorescence emitted by the marker protein with a microscope. The signal detected by these methods indicates the distribution of CapZ in the section.

  The aforementioned exogenous CapZ can be applied as CapZ to be introduced into the CapZ-TG animal. The CapZ may be CapZ cloned from the same species of animal or CapZ cloned from a heterogeneous animal. For example, the CapZ introduced into the mouse may be a CapZ derived from a mouse or a CapZ derived from another mammal such as a human, a rat, or a rabbit. Here, CapZ to be introduced has a function of localizing in spines and / or a function of binding to F-actin in nerve cells depending on signal input. As long as it has this function, deletion, substitution, or addition of one or several amino acid residues may be performed in the amino acid sequence of CapZ to be introduced.

The marker protein is not particularly limited, and conventionally known marker proteins can be applied, and examples thereof include alkaline phosphatase, HRP, GFP, EGFP and the like.
The peptide tag is not particularly limited, and a conventionally known peptide tag can be applied, and examples thereof include an HA tag, Myc tag, His tag, FLAG tag, and GST tag.
Detecting a signal derived from the marker protein and / or a signal derived from an antibody that specifically binds to the peptide tag is preferable because it becomes easier to examine the localization of the fusion protein containing the CapZ. .

When using the CapZ-TG animal, the expression of the fusion protein in the CapZ-TG animal corresponds to the period during which the learning behavior was performed, as much as possible, in order to amplify the response to the learning behavior being performed. It is preferable to induce with a narrow temporal width.
Specifically, the expression of the fusion protein is preferably controlled by a specific promoter. Examples of suitable promoters include Arc promoters, c-Fos promoters, and the like, whose expression is induced depending on neural activity input. In addition, a tetracycline-inducible promoter, a cre-dependent promoter structure, and the like that can control expression at a desired timing can also be exemplified as suitable ones.

  Among those exemplified, the Arc promoter is more preferable. By using the Arc promoter, for example, when a transgenic mouse into which CapZ has been introduced (hereinafter sometimes referred to as a CapZ-TG mouse) is allowed to perform learning behavior related to spatial recognition, the CapZ-TG mouse in the brain A signal is input to a neuron belonging to the ray layer of the CA1 field of the hippocampus, and the fusion protein can be expressed in the neuron with the input. That is, the expression of the fusion protein is triggered by causing the learning behavior to be performed in a neuron cell that is considered to accumulate memory regarding the learning behavior. This mechanism can be used to distinguish the distribution of newly expressed fusion protein from the endogenous CapZ that originally existed when accumulating memory regarding the learning behavior. That is, it becomes possible to distinguish between CapZ related to memory accumulated before performing the learning behavior and EGFP-CapZ (fusion protein) expressed by performing the learning behavior. As a result, it is considered that the distribution of the newly expressed fusion protein better reflects the role of CapZ as a learning marker in the accumulation of memory related to the learning behavior.

[Evaluation process]
The evaluation step in the evaluation method of the present invention is a step of evaluating the brain state based on observation of the localized state of the CapZ.
In the evaluation step, analyzing the distribution of signals derived from CapZ detected in the detection step, and when the CapZ is localized in some spines, the animal is in a state having normal learning ability, or The animal is evaluated and judged to have a normal learning ability at least in the learning process.
In addition, in the evaluation process, when the distribution of the signal derived from CapZ detected in the detection process is analyzed and the CapZ is not localized in some spines, the animal is not in a state having normal learning ability. (It is an abnormal state), or it is evaluated and judged that the animal is not in a state having a normal learning ability at least in a learning process (it was in an abnormal state).

  In the present specification, the ratio of spines in which the CapZ signal is strongly detected among the spines contained in the brain region to which signals are input in accordance with the learning behavior is referred to as “CapZ spine localization rate” or simply This is called “localization rate”. The localization rate is obtained by capturing the CapZ intensity in each spine defined by phalloidin staining from the entire image of the brain slice, and the intensity distribution (histogram) is not close to the maximum likelihood value, but prominently high. The intensity having a value can be defined by the ratio of the distribution.

Many spines are usually present in the observed nerve cells and brain regions. Here, “a part of CapZ is localized in the spine” means that the concentration of CapZ is not increased in all of the plurality of spines but the concentration of CapZ is increased only in some of the spines.
Recent studies suggest that changes in neuronal synaptic plasticity related to memory occur only in some neurons. According to the study by the present inventor, as described above, in the hippocampus of a brain that has passed a learning behavior in a wild-type mouse having normal learning ability and has passed for 24 hours, a nerve that forms a specific neural circuit The CapZ signal is strongly detected only in 1 to 5% of all spines of the cell group.

In the present invention, whether or not the animal has normal learning ability can be determined based on the localization rate. Specifically, after letting an animal perform a learning action, a predetermined percentage of the spines of nerve cells in a brain region that is considered to form a memory related to the learning action in a section of the brain at a predetermined time elapsed When the CapZ signal is strongly detected only in the spine, that is, when the localization rate is high, it can be determined that the animal has normal learning ability.
The predetermined time is preferably 4 hours to 72 hours, more preferably 8 hours to 48 hours, and even more preferably 12 hours to 24 hours. Within this range, it can be more reliably determined that the memory relating to learning behavior has been accumulated (formed) in the nerve cell.
The localization rate is preferably 0.01 to 10%, more preferably 0.1 to 10%, and still more preferably 0.1 to 5%. Within this range, it is possible to more reliably discriminate between the case where memory related to learning behavior is accumulated (when normal learning ability is possessed) and the case where memory is not accumulated (when normal learning ability is absent). .

In the evaluation method of the present invention, a disease model animal exhibiting a disease state can be used as the animal. Examples of the disease include Down's syndrome, schizophrenia, dementia, a disease exhibiting a known learning disorder, or a known mental illness. A well-known thing can be applied to such a disease model animal.
Further, as the disease model animal, a CapZ-TG animal into which a gene encoding the fusion protein is introduced may be processed so as to become a disease model animal. It is preferable to use this disease-modeled CapZ-TG animal because it becomes easier to detect CapZ in the animal. Hereinafter, “disease model animal” includes “disease modeled CapZ-TG animal” unless otherwise specified.

  The method for producing the disease model animal is not particularly limited. For example, a method for introducing the disease-related gene into a fertilized egg, a method for producing a knockout mouse lacking or replacing the disease-related gene by a conventional method, a disease-related gene A method of introducing into a nerve cell by a viral vector or electroporation, a method of administering a drug that acts on the brain and exhibits a disease-like symptom, a method of partially damaging the brain, or a stress that induces a disease-like symptom The giving method etc. are mentioned.

Examples of the disease-related gene include Down's syndrome, schizophrenia, dementia, a disease presenting a known learning disorder, or a gene associated with a known mental disease.
The disease model animal exhibits symptoms of the disease or symptoms close to the disease. In addition, the disease modeled CapZ-TG animal is an animal that exhibits symptoms of the disease or symptoms close to the disease, and into which the fusion protein has been introduced.
For example, it is known that a disease model mouse into which a chromosome containing a gene related to Down's syndrome has been introduced exhibits a learning disorder.
By using a disease model animal and a healthy animal, the state of the brain exhibiting a learning disorder or mental illness related to the disease and the state of the brain having normal learning ability are expressed in the distribution of CapZ (fusion protein). Comparisons can be made based on differences.

<Medicine screening method>
The pharmaceutical screening method of the present invention comprises a step of administering a test substance to the disease model animal exhibiting a learning disorder or a mental disorder, a learning step for causing the animal to perform learning behavior, A detection step for examining the localization of CapZ in a neuron, and an evaluation step for evaluating the state of the brain based on the localization of the CapZ. In the evaluation step, the CapZ is localized in some spines. In this case, the test substance is judged to be useful as a medicament for treating or preventing the learning disorder or mental illness.

The learning step in the pharmaceutical screening method of the present invention may be performed in the same manner as the learning step in the evaluation method of the present invention described above, except that a test substance is administered to an animal in advance.
The animal is preferably the above-mentioned disease model animal that exhibits learning disabilities or mental illness.

As a disease model animal to be used in a pharmaceutical screening method, it is suggested in advance that the memory related to the learning behavior is not normally accumulated or the learning memory is not normally established even if the learning behavior is performed. Or what has been confirmed is preferable. Furthermore, it is preferable that the disease model animal has been confirmed in advance that CapZ is not normally localized in a brain region related to learning memory even when the learning behavior is performed.
In addition, as the disease model animal, even if the learning behavior is performed, it is suggested or confirmed in advance by another method that the memory related to the learning behavior is not normally accumulated or the learning memory is not normally established. More preferably, it is confirmed in advance that CapZ is not normally localized in the brain region related to the learning memory when the same learning behavior is performed. However, in this method, there is a possibility that the result is different from the determination of the effect in the conventional learning test. For example, during a fear conditioning test, animals that cannot move due to abdominal pain with the test substance may be misinterpreted as having a freezing reaction even if they are not remembered, or even though they remember fear According to the pharmaceutical screening method of the present invention, even if the judgment is incorrect in the conventional memory test, such as when it is judged that it is not memorized because it moves in a confusion (not showing a freezing reaction) Please note that it may be possible to judge accurately. Special attention should be paid to models of schizophrenia and depression, in which cognitive impairments such as memory tend to be masked by other symptoms such as dullness (malaise).

As the test substance, those having low toxicity with respect to disease model animals are preferable, and those expected to improve the disease are more preferable.
The dose and administration method of the test substance are not particularly limited, and may be administered by a known method with a pharmacologically acceptable dose.

  The detection step in the pharmaceutical screening method of the present invention may be performed in the same manner as the learning step in the aforementioned evaluation method of the present invention. It is preferable to use the disease-modeled CapZ-TG animal because the detection of CapZ becomes easier by using a marker protein or peptide tag bound to CapZ.

  The evaluation step in the pharmaceutical screening method of the present invention is a step of evaluating the state of the brain of the disease model animal to which the test substance has been administered based on the localization of the CapZ.

The evaluation can be performed in the same manner as the evaluation method according to the present invention described above. The CapZ-TG animal shows the CapZ localization state (localized in some spines) as a normal indicator, and the CapZ of the disease model animal before the test substance is administered Localized state (not localized in some spines) is used as an index of abnormality, and when the localized state of the CapZ indicated by the disease model animal to which the test substance is administered approaches from normal to normal, or the test substance is When the localization rate of the CapZ indicated by the administered disease model animal increases, it can be evaluated that the learning ability of the disease model animal that has been abnormal has been restored to normal or has become close to normal.
Based on this evaluation, it can be judged that the test substance is useful as a medicine for treating or preventing learning disorders or mental disorders exhibited by the disease model animals.

In the evaluation step of the pharmaceutical screening method, when the CapZ detected in the previous detection step is localized in some spines, the abnormal learning ability of the disease model animal has been restored to normal or is normally The test substance is evaluated as approaching, and it is determined that the test substance is useful as a medicine for treating or preventing a learning disorder or a mental disorder exhibited by the disease model animal.
Further, in the evaluation step, when the localization rate of the CapZ detected in the detection step in the previous stage is increased, it is evaluated that the learning ability that was an abnormality of the disease model animal has been restored normally or is close to normal, It is also possible to determine the degree of the effect of the test substance that treats or prevents the learning disorder or mental disorder exhibited by the disease model animal.

<Relationship between the present invention and conventional memory research>
In order to exert high-order functions of the brain such as memory, each nerve cell must have individuality and work in a shared role. If the majority of neurons act in synchrony without sharing their roles, they should have convulsions such as epileptic seizures. However, in previous research on memory, there was no method for analyzing which part of which neuronal cell is changing and how it works.

Increased concentration of CapZ (immunohistological staining intensity) at the site of long-term enhancement after artificial stimulation of a part of the brain to cause long-term enhancement, and spines with high staining intensity rather than being uniformly present in all spines Has already been reported by the inventors in the following paper (6).
Genes Cells. 2010 Jun; 15 (7): 737-47. Epub 2010 Jun 10. “Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.” Kitanishi T, Sakai J, Kojima S, Saitoh Y, Inokuchi K, Fukaya M, Watanabe M, Matsuki N, Yamada MK.
However, there were two problems in the experiment of this paper. 1. Since the stimulus is artificial, there is no guarantee that it is the same as the input related to learning. 2. In the case of immunohistochemical staining, there is a problem of antibody permeability, and it is known that there are molecules that are not stained even if present in the post-synaptic region. Therefore, the present inventor prepared a transgenic mouse (CapZ-TG mouse) expressing CapZ and EGFP as a fusion protein as a method different from immunostaining, and localized CapZ with the natural activity of the brain. It was found that it could be detected. Specifically, let the CapZ-TG mouse perform learning behavior (context-dependent fear conditioning), create a brain section 24 hours after memory should have been fixed, and associate memory of context and fear Observing the dorsal hippocampus necessary for the study reveals a characteristic pattern compared to the brains of animals that are not learning. It is a pattern in which some spines in the dorsal hippocampal CA1 field have EGFP intensity that exceeds the normal dispersion range (conspicuously high) compared to others. It was also found that there are dendrites with many such spines. The result of this transgenic mouse expressing EGFP-CapZ fusion protein denied the possibility that there was a difference in the abundance between spines due to the permeability of the antibody, which was a problem in immunohistochemical staining. .

In the first place, the transmission of information between neurons is thought to be carried by a small number of neurons. The basis for this is the experimental fact that only about 10% of neurons are active in any learning behavior regarding the generation of action potentials of neurons (Henze et al. JNP). Assuming information processing by such a small number of neural activities, it is necessary for a functional neural circuit to form a state in which information input propagates through only a certain part, such as water flowing through a river. In order to accumulate the memory related to learning, it is necessary that a part of the nerve connection is different from the surroundings, and a part that easily receives a new input and a part that easily flows water are generated. The change spreads if it is not a “few”.
From past experiments by the inventors, it is known that the concentration of CapZ increased in a long-term potentiating site (a site where the nerve transmission efficiency was increased) in an experiment that artificially stimulated long-term potentiation. By analogy, there is a high probability that spines with a high EGFP-CapZ strength have undergone long-term potentiation and coding (formation of a part where stored information can easily flow). In addition, if there is a dendrite in which the spine is accumulated, the nerve cell can work as a coding neuron. The frequency of appearance of spines with high EGFP-CapZ intensity found by the present inventor is small, and it is reasonable in view of the small number of neurons that are active in relation to specific memory.
(Reference: J Neurophysiol. 2000 Jul; 84 (1): 390-400. “Intracellular features predicted by extracellular recordings in the hippocampus in vivo.” Henze DA, Borhegyi Z, Csicsvari J, Mamiya A, Harris KD, Buzsa ' ki G.)
In the evaluation method of the present invention, when the localization ratio of CapZ localized to some spines is high beyond the normal dispersion range, it is defined as a brain that has been learned and used for evaluation of brain function. .

<Example 1>
[Preparation of non-human animals]
As a non-human animal, a transgenic mouse (hereinafter referred to as CapZ-TG mouse) was prepared and used. CapZ-TG mice were produced in order to express CapZ fused with EGFP by the following method. The transgene was prepared by sequentially recombining DNA sequences encoding the following DNA sequences or amino acid sequences (1) to (8) by genetic recombination (FIG. 1).
(1) mouse Arc promoter (SmaI-NaeI fragment; 7.1 kb)
(2) Tetracyclin-responsive transcriptional activator (Clontech; derived from pTet-off)
(3) P2A sequence (TSATNFSLLKQAGDVEENPGPVAT; artificial synthesis)
(4) EGFP (Clontech; derived from pEGFP-N1)
(5) Spacer (SGRTQISSS; artificial synthesis)
(6) HAtag (YPYDVPDYA; artificial synthesis)
(7) mouse CapZ beta 2 full length 272a.a. (Met-Cys) (isolated by RT-PCR; Genbank Accession No. NM_009798; Mus musculus capping protein (actin filament) muscle Z-line, beta (Capzb), transcript variant 2, mRNA)
(8) SV40-polyA (Clontech; derived from pEGFP-N1)

The mouse Arc promoter in (1) is a BAC clone (B6Ng01-232C07, etc.) containing the relevant part of the mouse genome (between Ch15 and 74.50M-74.51M), and the BAC library (http://dna.brc.riken.jp). /ja/MSMbac.html etc.) and can be cloned from around 7.1 kb of digested SmaI-NaeI.
The P2A sequence in (3) is a signal to synthesize equal amounts of two different proteins (in this case (2) and (4)-(7) fusion proteins) from a series of cDNAs (Donnelly et al. J Gen Virol 2001, 82: 1013-1025.). Note that (2) is a transcription factor in preparation for the future expression of another gene in this CapZ-TG mouse.
(7) CapZ is a dimer of alpha and beta, and beta has 1 and 2 splicing variants. Since beta2 is the main in the brain, this is used.

The transgene prepared above was linearized using a restriction enzyme while removing the vector portion, and then purified by the phenol chloroform method and ethanol precipitation. Furthermore, gel electrophoresis was performed, a band of the corresponding length was cut out so as not to contain the vector portion, and purification using a Qiagen gel extraction kit was repeated twice to highly purify.
The purified transgene was injected into the nucleus of the fertilized egg, and a normally developed embryo was borrowed and transferred to the abdomen. Among 42 pups, 7 lines were identified as CapZ-TG mice. Since the number of copies introduced for each line was (1, 3, 10, 10, 10, 10, 100), 100 lines were used for the experiment.

  Since the produced CapZ-TG mouse has an Arc promoter, EGFP-HA-CapZ fusion protein can be selectively expressed in recently activated neurons.

[The process of performing learning behavior]
The prepared CapZ-TG mice were subjected to contextual fear conditioning as a learning behavior by the following method.
Ohara Medical Industry's contextual fear conditioning device (shock generator model number: SGA-2010, experimental chamber: model number CL-3002) and soundproof box (model number CL-4210C) were used. First, the mouse was placed in the chamber of the soundproofed device for 5 minutes. Of these 5 minutes, a total of 2 electric shocks (0.2 mA, 2 seconds) were applied to the mouse paw, once at 1 minute and once at 4 minutes. The state of electric shock was observed through a camera and confirmed. After a 5 minute conditioning session, the mice were returned to their normal cage.
After this learning behavior, it is generally known that mice that receive an electric shock learn and memorize context-dependent fear conditions that link "being placed in a specific chamber" and "being subjected to an electric shock" However, the mouse is not tested for memory.
Separately from this step, it has been separately confirmed by a conventional method that the CapZ-TG mouse can learn and memorize the context fear condition by performing a test after 24 hours. That is, in this confirmation, there was no significant difference between the CapZ-TG mouse and the wild type mouse that normal learning ability was exhibited (see FIG. 14). In the test, mice were placed in a shocked chamber for 5 minutes in accordance with a conventional method, and the percentage of time during which freezing, a fear reaction, occurred was recorded. The recording was counted as Freezing when the mouse part (black) of the image taken every 0.5 seconds from the camera at the top of the chamber did not cause a constant shift for 2 seconds. Images were acquired by the Ohara Medical Industry equipment and transferred to a PC, and then automatically measured with the software TimeFZ1.

[Detection process to investigate the localization of CapZ in neurons in the brain of CapZ-TG mice]
CapZ-TG mice 24 hours after contextual fear conditioning are anesthetized, the needle is inserted into the left ventricle within 30 seconds (usually within 10 seconds) after thoracotomy, 2 ml of PBS, and then 0.1M phosphorus containing 2% paraformaldehyde Acid buffer (both ice-cold) 20-30ml (at approximately 100mmHg that does not exceed the mouse's systolic blood pressure) systemically perfused and fixed, remove the brain and halve the midline, and for another hour, After fixing in a 4 ° C room with sufficient shaking, it was fixed. The fixed mouse brain was coronally cut with a vibratome. For the following observations, the CA1 ray layer of the dorsal hippocampus (in the range of coordinates Bregma 1.4mm-2.2mm) and the inside (lateral, within 1.5mm) were used for spatial learning. In the following, the results for the relevant part of the CA1 radiation layer are shown, but the same tendency was observed in other parts (reticular molecular layer, polymorphic layer) in CA1 and CA3. In addition, protein will be in the state which does not change, such as decomposition | disassembly and movement, by a fixing process. The fluorescence of the fluorescent protein EGFP is not lost by this fixation method.

The obtained sections were treated with 0.1% TritonX100 for 15 minutes, treated with 5% goat serum to prevent nonspecific adsorption for 30 minutes, and then diluted with PBS containing 1% goat serum. Primary antibody (1: 3000, chicken IgY, manufactured by Phosphosolutions; model 1100) was reacted at 4 ° C. with shaking overnight. After the reaction, it was washed 3 times with PBS. Next, as a secondary antibody, fluorescence (Alexa647) -labeled anti-chicken IgY (1: 1000, manufactured by Invitrogen; model number A21463) was diluted in PBS containing 1% goat serum and reacted with the sections simultaneously with Rhodamine-phalloidin. (0.7 unit / ml, manufactured by Invitrogen; model R415) was added and reacted at room temperature for 1 hour or at 4 ° C. for 3 hours to perform double staining with phalloidin and anti-MAP2 antibody.
The antibody solution was centrifuged (15,000 × g, 5 minutes) before the reaction, and care was taken not to cause pseudo-staining with an antibody that was denatured and aggregated by using the supernatant.
The stained section was observed with the laser confocal microscope: Olympus FV1000 (inverted microscope: IX81, objective lens: UPLSAPO 100 × ONA1.40) at the slowest scan speed, and the photoelectron gain was suppressed to within 660V. Images were acquired with a double tube. At the time of image acquisition, the laser intensity was adjusted according to the brightest sample so that the maximum intensity that could be imaged was not exceeded. The results are shown in FIGS.

The processing method of the acquired image is as follows.
<Image processing method>: Intensity based on the maximum and minimum values of the pixel intensity in the image according to the widest value in the image to be compared so that there is no substantial cut value when processing the image Was enlarged. For example, if there are two images of 10-210 and 8-150 in an 8-bit image of 0-255 gradation, both 8-210 are extended to 0-255. In addition, in principle, original drawings were published for the displayed figures, and median filters were applied to make the figures 2 to 6 and 11 to 13 obtained at 4 times zoom easy to see.
The median filter is generally used to eliminate flicker noise derived from a measuring instrument, and is based on the principle that an intermediate value of surrounding points is adopted as a median value to make a prominent value. Here, since the radius is set to 1 pixel, 3 × 3 points are used.

  FIG. 2 shows the EGFP-derived green fluorescence signal of EGFP-CapZ. As shown in FIG. 2, the EGFP-CapZ signal in the radiation layer of the hippocampal CA1 area of CapZ-TG mice after learning behavior is localized in part. The bar in this figure is 2 micrometers, and it can be seen that the portion where the signal is detected is up to about 1 micrometer in diameter and has a variation in size.

  FIG. 3 shows spines stained with red fluorescence of Rhodamine-phalloidin in the same field of view as FIG. Spine is thought to be stained with Rhodamine-phalloidin because it contains a lot of fibrous actin. On the dendrites of excitatory neurons in the brain, there is a dense projection structure called spine (spinous process) with a head diameter of about 1 micrometer and varying in size. Is known. The spine is the part of the synapse that receives the excitatory signal. When information to be stored by a nerve cell is received by a certain spine, the spine should change to make it easier to convey information than other spines in order to respond to the next recall stimulus. That is, a part where some change occurs with the formation of memory must be present in some spines.

  The left of FIG. 4 shows the same EGFP-CapZ signal as in FIG. 2, and the right of FIG. 4 shows the same Rhodamine-phalloidin staining as in FIG. Both were acquired simultaneously as separate fluorescent signals from the same field of view. Since the arrows are at the same position, it can be clearly seen that the signal of EGFP-CapZ (left) is strongly localized only in a part of the spine stained with Rhodamine-phalloidin (right).

  FIG. 5 shows a dendrite in which MAP2 is present superimposed on the signal of EGFP-CapZ in FIG. A granular EGFP-CapZ signal is present along the streak dendrites as spines protruding from the dendrite. This figure further reinforces that the EGFP-CapZ signal is in the spine. In addition, although there seems to be a gap between the dendrite and the spine, this is considered to be because CapZ does not exist in the neck portion of the spine. There is a mushroom type spine, which has a thin neck-like structure called a neck. Some necks are longer than 2 micrometers (the length of the bar shown in FIG. 5).

  FIG. 6 shows only dendrites in which MAP2 is present in the same visual field as FIG. 5 except for the signal of EGFP-CapZ, for reference when interpreting FIG. The part that looks round is not a spine, but seems to be a cut surface of a thin dendrite toward the front of the screen.

From the above observations, it is clear that there are places where the concentration of EGFP-CapZ is locally high in the nerve cells in the brain part related to the learning of the mouse brain subjected to contextual fear conditioning. The site where EGFP-CapZ mainly exists at a locally high concentration is considered to be a spine because it is stained with Rhodamine-phalloidin. Further, the signal intensity of EGFP-CapZ is not high in all the spines observed in the brain region, but the signal intensity of EGFP-CapZ is high only in some spines. In other words, it is understood that CapZ is localized in some spines in the mouse brain in which learning behavior is performed (context fear conditioning is performed).
Of the spines observed in the brain region, the proportion (localization rate) of spines in which the signal intensity of EGFP-CapZ was remarkably increased was estimated to be 1 to 5%.

  FIG. 7 is a specimen prepared under the same conditions as the brains of FIGS. That is, it shows the EGFP-CapZ signal from the ray layer of hippocampal CA1 in the brain fixed 24 hours after letting CapZ-TG mice perform learning behavior (context fear conditioning). The scale bar is 10 micrometers, and it can be seen that the portion where EGFP-CapZ exists is sparse even when a wide range of signals is taken.

<Comparative Example 1>
Using the above CapZ-TG mice, mice were treated in the same manner as in Example 1 without contextual fear conditioning. The obtained sections were observed in the same manner. The results are shown in FIGS.

  FIG. 8 shows the distribution pattern of EGFP-CapZ in the radiation layer of the hippocampal CA1 area of the mouse brain in which learning behavior was not performed. FIG. 9 is a photograph in which the overall intensity of the same image as FIG. 8 is lowered. FIG. 8 shows that even if the image is taken so that the intensity in FIG. 8 is lowered, the image in FIG. That is, comparing FIG. 7 of Example 1 and FIGS. 8 to 9 of Comparative Example 1, FIG. 7 differs from FIGS. 8 to 9 in that “CapZ is localized to“ part of spine ”. It is clear. Here, “CapZ is localized in“ some ”spines” means that the intensity of signals derived from CapZ detected by spines is stronger than that of dendritic trunks (other than spines). At the same time, it means that there is a bias in the intensity of the signal between spines in the same region (a specific brain region, here the dorsal hippocampus involved in spatial memory), Conceivable. In addition, since the signal intensity of EGFP varies depending on, for example, pH (mainly in the fixing solution) and the observation depth, discussion on comparison of absolute intensity is difficult at this time.

  The left of FIG. 10 is a photograph of phalloidin staining (spine staining) in the same visual field as FIG. 7, and the right of FIG. 10 is a photograph of phalloidin staining (spine staining) in the same visual field as FIG. It can be seen that there is no change in the spine density. In other words, the bias in the signal derived from CapZ detected in FIG. 7 is not due to the disappearance of the spine itself, but because the concentration of CapZ varies depending on the amount of CapZ for each spine. It is done.

  Based on the above observations, EGFP-CapZ appears to be present in the entire spine at the same concentration in neurons of the mouse brain that did not perform learning behavior (no contextual fear conditioning), and other spines in other concentrations. Some spines that were slightly higher than those were not found.

The reason why the EGFP-CapZ signal distribution when learning behavior is performed appears to disappear without leaving some spines as described above. This can be interpreted as being decomposed. As a finding supporting this, it is known that there is a degradation signal in the CapZ molecule, and if the binding proteins BAG3 and Hsc70 do not coexist, they are degraded by the proteasome independent of ubiquitin.
(Reference: Circ Res. 2010 Nov 12; 107 (10): 1220-31. BAG3 and Hsc70 interact with actin capping protein CapZ to maintain myofibrillar integrity under mechanical stress.Hishiya A, Kitazawa T, Takayama S.)

  From the results of Example 1 and Comparative Example 1, the memory accumulated in the mouse brain as a result of the learning action and the EGFP-CapZ newly expressed in the mouse brain by the learning task were localized in some spines. The fact that it was detected (that it was detected strongly biased to some spines) indicates a high correlation. That is, the fact that EGFP-CapZ was selectively localized in a part of the spine of the nerve cell related to the learning behavior indicates that the memory related to the learning behavior was recorded in the spine. it is conceivable that.

<Example 2>
Wild-type mice were used instead of CapZ-TG mice, and anti-CapZ beta2 antibody (1: 300, mouse monoclonal IgG, manufactured by The Developmental Studies Hybridoma Bank; model number 3F2.3-s, instead of EGFP fluorescence) An antibody against the C-terminal sequence part that exists only in Valiant2 of beta is reacted simultaneously with the anti-MAP2 antibody, and fluorescence (Alexa488) -labeled anti-chicken IgG (1: 1000, manufactured by Invitrogen; model number A11029) as the secondary antibody ) Was used in the same manner as in Example 1 except that it was used for detection. The result is shown in FIG. It has been confirmed that the anti-CapZ beta 2 antibody used shows very high specificity in brain proteins in immunoblotting experiments (single band in supplementary figure 1 of Non-Patent Document 4).

  In FIG. 11, the white area indicates the signal derived from the anti-CapZ antibody. The bar is 1 micrometer, and it can be seen that a signal of almost the same size as that seen in FIG. 2 is obtained. In addition, it has been confirmed that this signal site is localized in a part of the spine, as shown in FIGS. This observation led to the same conclusion as in FIG. That is, it was found that a strong CapZ signal was observed in some spines even by the method of immunostaining in the wild type.

<Comparative Example 2-1>
Mice were treated in the same way as Example 2 without contextual fear conditioning. The obtained sections were observed in the same manner. The result is shown in FIG.
In FIG. 12, the white area indicates a signal derived from the anti-CapZ antibody. As a result of this observation, a relatively uniform signal is observed in the mouse brain in which the learning behavior is not performed (the context fear conditioning is not performed) as in FIG. In other words, the phenomenon that CapZ was localized in some spines was not found.

<Comparative Example 2-2>
Down's syndrome model mouse (homologous to human 21st chromosome, which has three due to Down's syndrome, part of mouse chromosome 16 is added to make it partially partial trisomy), conducted using Ts1Cje mouse Contextual fear conditioning similar to Example 2 was performed and analyzed as in Example 2. The result is shown in FIG.
The mouse used was provided by Dr. CJEpstein, the creator, and then provided by Dr. Kazuhiro Yamakawa of RIKEN. The following references have reported that this mouse exhibits learning disabilities.
(Reference: Proc Natl Acad Sci US A. 1998 May 26; 95 (11): 6256-61. “Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities.” Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, Epstein CJ, Huang TT.)

In FIG. 13, the white area indicates the signal derived from the anti-CapZ beta2 antibody.
In this observation, in the brain of Down syndrome model mice with learning disabilities, despite the learning behavior, the concentration of CapZ was measured to be high overall, but the signals detected by each spine were less biased, A strong signal was not detected only in some spines.

  The method for evaluating the learning ability of a non-human animal of the present invention can provide an objective index as to whether or not the animal has learning ability. This evaluation method can be used to evaluate the effect of drugs administered to the animals on learning ability. Therefore, the evaluation method of the present invention can be used when screening for drugs useful for the treatment or prevention of learning disorders and mental disorders.

Claims (7)

A method for evaluating whether a non-human animal is in a state of having normal learning ability,
A learning step for causing the animal to perform a learning action;
A detection step of examining the localized state of CapZ in nerve cells of the animal brain;
Based on the observation of the localized state of the CapZ, an evaluation step for evaluating the state of the brain,
A method for evaluating the learning ability of a non-human animal, comprising: In the evaluation step,
The evaluation method according to claim 1, wherein when the CapZ is localized in a part of the spine, the animal is evaluated as having a normal learning ability. The non-human animal is a transgenic animal into which a gene encoding a fusion protein in which CapZ and a marker protein and / or a peptide tag are bound is introduced,
The localization of CapZ is examined by detecting a signal derived from the marker protein and / or a signal derived from an antibody that specifically binds to the peptide tag. Evaluation method. Expression of the fusion protein in the transgenic animal,
The evaluation method according to claim 3, wherein the input is induced by the input in a brain region that receives the input in accordance with the learning behavior. The evaluation method according to any one of claims 1 to 4, wherein the animal is a disease model animal. CapZ is protein of the following (i) or (ii), The evaluation method as described in any one of Claims 1-5 characterized by the above-mentioned.
(i) A protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5.
(ii) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and spine depending on input to a neuron; A protein that has a function localized in. Administering a test substance to the disease model animal according to claim 5, which exhibits a learning disorder or a mental disorder;
A learning step for causing the animal to perform a learning action;
A detection step for examining the localization of CapZ in neurons of the animal brain;
An evaluation step for evaluating the state of the brain based on the localization of the CapZ;
Including
In the evaluation step,
When CapZ is localized in some spines, the test substance is judged to be useful as a drug for treating or preventing the learning disorder or mental illness.