JP2016095299A - Screening method of curative medicine for posttraumatic stress - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a screening method of a substance to be inspected, to curing of a posttraumatic stress in which forgetfulness of memory is an index.SOLUTION: A screening method of a curative medicine for posttraumatic stress comprises: a first step for, in a state that terror memory in a non-human animal which is subjected to a terror condition-related context learning task, is converted into memory which does not depend on a hippocampus, deriving gene expression in the hippocampus by recalling the terror memory; and a step for, after the first step, administering a substance to be inspected for promoting formation of nerve in the hippocampus, to the non-human animal, in which an affection which is given to the non-human animal by the substance to be inspected, is detected, in which forgetfulness of the terror memory in the non-human animal serves as an index.SELECTED DRAWING: Figure 12

Description

  The present invention relates to a screening method for a therapeutic agent for post-traumatic stress disorder using forgetting memory as an index, using a non-human animal subjected to a fear conditioning context learning task. More specifically, a first step that recalls fear memory for non-human animals and restores hippocampal dependence, and a test substance that promotes neurogenesis in the hippocampus is administered to non-human animals after this first step. And a second step of screening a therapeutic agent for post-traumatic stress disorder.

In modern societies where there are many anxiety factors such as interpersonal relationships, occupations, living environment, economic problems, etc., “mental illness” represented by psychosomatic disorders and posttraumatic stress disorder (PTSD) is a kind of It is a social problem, and the number of people suffering from these diseases is increasing year by year. The post-traumatic stress disorder (PTSD) is said to have a lifetime prevalence of about 10%, and WHO predictions suggest that the number of patients will increase dramatically in the future.

  Post-traumatic stress disorder (PTSD) is caused by experiencing shocking events related to life and death such as natural disasters such as earthquakes, traffic accidents, sexual damage and domestic violence. It is a mental disorder that develops due to traumatic trauma (trauma) due to shocking events, that is, trauma memory (fear memory) of fear experiences. The main symptoms of post-traumatic stress disorder are (1) intrusive symptoms such as flashback and re-experience, (2) paralysis such as avoidance from things related to trauma and emotional atrophy, (3) There are hyperwakening such as sleep disorder and excessive startle response.

  As a treatment for this post-traumatic stress disorder (PTSD), prolonged exposure therapy (PE) is internationally recommended. This continuous exposure therapy (PE) is a cognitive behavioral therapy that reminds you of the shocking events that have caused traumatic trauma and gradually gets used to the fear of traumatic trauma. Ultimately, it is a cure that learns that there are no dangers of recalling shocking events, and that words can overcome trauma.

  In recent years, it has been believed that the development of post-traumatic stress disorder (PTSD) is closely related to fear memory due to the shocking event that caused it. Since the fear memory is formed in the brain, it is considered that failure of fear memory control in the brain is greatly related to the development of post-traumatic stress disorder (PTSD).

Here, the process of memory formation in higher animal species including humans will be described. These species acquire unstable short-term memory that can only be temporarily stored by experiencing some event. This short-term memory is converted into long-term memory that is retained in the brain for a long time through a process called “immobilization” through expression of a gene group typified by an early response gene in the hippocampus.
Further, in the process of recalling memory, it is shown that the long-term memory that is fixed and held is returned to an unstable state as in the case of short-term memory. There is a process called “re-immobilization” that re-stores such unstable memory after recall in the brain. This “re-immobilization” of the memory is similar to “immobilization” and gene expression in the hippocampus. Is known to be necessary.
And the memory formed in the brain is recalled depending on the hippocampus for a certain period after this memory is formed, while the “old (old) memory” stored in the brain beyond that period "Is known not to depend on the hippocampus but to the cerebral cortex. On the other hand, in the case of fear memory, if the state of just remembering the fear memory without maintaining the fear experience is maintained, it is remembered that there is no need to feel fear in response to the fear memory, and the fear memory is suppressed. "Erase" of memory occurs. This “erasure” is known to be the biological basis of continuous exposure therapy (PE).

On the other hand, the inventor of the present invention has analyzed the stability of fear-conditioned memory in a mouse (Non-patent Document 1). In this analysis, the Contextual Fear Conditioning Test was used. In this Contextual Fear Conditioning Test, first, as a training (fear conditioning), wires are placed on the floor, and the mouse is placed in a chamber that becomes a conditioned stimulus (CS) for 3 minutes. The electric shock which becomes (unconditioned stimulus; US) was given, and fear memory was formed.
In order to analyze the stability of recent memory, that is, memory that is recalled depending on the hippocampus, 24 hours after the training, the mouse was returned to the same chamber again for 3 minutes to recall fear memory (re-establishment). Exposure). Immediately before this re-exposure, a protein synthesis inhibitor anisomycin (ANI) was administered intraperitoneally to observe whether fear memory was destroyed.
As a result, in the case of such recent memories, the destruction of the memories was observed by returning to the chamber for 3 minutes. On the other hand, to analyze the stability of the old memory, that is, the memory recalled without depending on the hippocampus, 8 weeks after the training, the mouse was returned to the same chamber again for 3 or 10 minutes to recall the fear memory. (Re-exposure). Immediately before this re-exposure, a protein synthesis inhibitor anisomycin (ANI) was administered intraperitoneally to observe whether fear memory was destroyed.
As a result, when the chamber was returned to the chamber for 3 minutes, destruction of fear memory due to protein synthesis inhibition was not observed. On the other hand, when it was returned to the chamber for 10 minutes and reminded of fear memory for a long time, destruction of fear memory was observed. From this analysis, it has been clarified that “re-fixation” of memory is induced by recalling old fear memories for a long time.

  In addition, the present inventor used the passive avoidance reaction task to apply the electric shock when moving the mouse from the light box to the dark box to form fear memory, and then influence the effect of returning the mouse to the light box. Analyzed (Non-Patent Document 2). Through this analysis, the present inventor has revealed that fear memory is enhanced by going through a process of “re-fixation” of memory after recalling memory. That is, it was clarified that “re-fixation” of memory contributes to enhancement of memory.

From the elucidating facts regarding the formation process of these memories, the cause of the post-traumatic stress disorder (PTSD) mentioned above is the cause of “excessive memory re-fixation” and the fear of fear memory that makes fear memory more and more intense. It is thought that it can be regarded as "abnormality of fear memory erasure" that cannot be done. For this reason, as a treatment method for post-traumatic stress disorder (PTSD), a drug administration targeting “re-fixation” or “erasure” of memory has been devised. For example, D-cycloserine, which is an NMDA receptor agonist, is known to promote “erasure” of memory. From this fact, the therapeutic method using the continuous exposure therapy (PE) in combination with the administration of D-cycloserine is known. Research is underway.

  On the other hand, recently, it has been clarified that nerve cells are newly produced in the hippocampal dentate gyrus of the adult brain (hereinafter referred to as “neurogenesis”). It has been suggested to develop new treatments. However, the role of neurogenesis in the brain has not been clarified.

In contrast, the present inventor used memantine, a non-competitive inhibitor of NMDA-type glutamate receptors, known to increase neurogenesis in the hippocampal dentate gyrus, and neurogenesis for memory formation ability in the brain. Was detected (Non-patent Document 3).
As a result, it became clear for the first time that administration of the memantine promoted neurogenesis in the hippocampus and that young neurons that had just matured contributed to the improvement of memory formation ability.

Suzuki et al., `` Memory Reconsolidation and Extinction Have Distinct Temporal and Biochemical Signatures '' The Journal of Neuroscience May 19,2004 Fukushima et al., `` Enhancement of fear memory by retrieval through reconsolidation '' eLife June 24, 2014 Ishikawa et al., “Time-dependent enhancement of hippocampus-dependent memory after treatment with memantine: Implications for enhanced hippocampal adult neurogenesis.” Hippocampus 2014 Jul; 24 (7): 784-93

  As described above, it is believed that fear memory due to the shocking event that caused the disease is closely involved in the development of post-traumatic stress disorder (PTSD). For this reason, easily detecting a therapeutic agent for post-traumatic stress disorder (PTSD) that can promote “forgetting” fear memory is a fundamental therapeutic agent for post-traumatic stress disorder (PTSD). This is a very important issue in order to reduce the labor and cost involved in the development of therapeutic drugs and the development of therapeutic drugs.

  In addition, as described above, as a treatment method for post-traumatic stress disorder (PTSD), research has been conducted on a treatment method using D-cycloserine in combination with the continuous exposure therapy (PE). However, in the process of continuous exposure therapy (PE), it is not possible to determine whether fear memory is about to be re-fixed or whether fear memory disappears. Further, there is a view that the D-cycloserine enhances fear memory when administered when the memory is about to be re-immobilized, and on the other hand, worses post-traumatic stress disorder (PTSD). In view of these problems, it is necessary to determine the timing of D-cycloserine administration in the treatment method in which administration of D-cycloserine is used in combination with the continuous exposure therapy (PE). There was the possibility of delaying the treatment of post-traumatic stress disorder (PTSD).

  Therefore, the present invention mainly provides a screening method for a test substance for treatment of post-traumatic stress disorder using forgetting memory as an index, using a non-human animal subjected to a fear conditioning context learning task. With a purpose.

As a result of diligent study for solving the above-mentioned problem, the present inventor recalled horror memory to a degree that induces gene expression in the hippocampus for non-human animals subjected to a fear-conditioning context learning task. By subjecting a human animal to the process of promoting neurogenesis in the hippocampus, it has been newly found that even if it is an old fear memory that has lost its hippocampal dependence after a long period of storage, it is forgotten. It came to complete.
That is, the present invention recalls the fear memory for the non-human animal in a state where the fear memory in the non-human animal subjected to the fear conditioning context learning task is converted into a hippocampal-independent memory. A first step of restoring hippocampal dependence, that is, inducing gene expression in the hippocampus, and a second step of administering a test substance that promotes neurogenesis in the hippocampus to the non-human animal after the first step; And a method for screening a therapeutic agent for post-traumatic stress disorder (PTSD), wherein the influence of the test substance on the non-human animal is detected using forgetting fear memory in the non-human animal as an index Is.
In this screening method, the first step can be performed 8 weeks after the non-human animal is subjected to a fear-conditioned context learning task.
Furthermore, in the first step of the screening method, the non-human animal can be returned to the chamber used for the fear conditioning context learning task for 10 minutes.
In this screening method, the gene can be an early response gene.
Furthermore, in this screening method, the non-human animal can be a rodent.

  According to the present invention, when used in combination with the continuous exposure therapy (PE), it is possible to easily perform an assay for a therapeutic agent that can achieve treatment promotion for post-traumatic stress disorder (PTSD). Further, when administered at the time of induction of memory re-fixation, it is not necessary to consider re-fixation or erasure of memory for D-cycloserine that can enhance fear memory, that is, it is not necessary to consider the timing of administration. It is possible to easily test for therapeutic agents for post-traumatic stress disorder (PTSD). Furthermore, according to the present invention, since it is possible to easily test a therapeutic agent for post-traumatic stress disorder (PTSD), it is possible to reduce labor and cost for developing the therapeutic agent.

It is a figure which shows the process of the fear condition context task (Contextual Fear Conditioning Test) used in order to show the relationship between the forgetting of memory and memantine (Experimental Example 1). It is a drawing substitute graph which shows the result of Experimental example 1 of FIG. It is a figure which shows the process of the fear condition context task (Contextual Fear Conditioning Test) used in order to show the relationship between forgetting of old memory and memantine (Experimental example 2). It is a drawing substitute graph which shows the result of Experimental example 2 of FIG. It is a figure which shows the process of the fear conditional context task (Contextual Fear Conditioning Test) used for examination of the novel gene expression induction in the re-fixation of old fear memory (Experimental example 3). 6 is a drawing substitute graph showing the results of Experimental Example 3 in FIG. 5. It is a figure which shows the process of the fear conditioning context task (Contextual Fear Conditioning Test) used for examination of the necessity of the novel gene expression in the re-fixation of old fear memory (Experimental example 4). It is a drawing substitute graph which shows the result of Experimental example 4 of FIG. It is a figure which shows the process of the fear condition context task (Contextual Fear Conditioning Test) used for examination of the hippocampal dependence revival concerning the old memory recalled for a long time (Experimental example 5). 10 is a drawing substitute graph showing the results of Experimental Example 5 shown in FIG. 9. It is a figure which shows the process of the fear conditional context task (Contextual Fear Conditioning Test) used in order to show the relationship between forgetting of old memory and memantine (Experimental Example 6). 12 is a drawing substitute graph showing the results of Experimental Example 6 in FIG. 11. It is a figure which shows the process of the fear conditioning context task (Contextual Fear Conditioning Test) used for examination of the reimmobilization induction | guidance | derivation of hippocampal dependence fear memory, and the relationship with memantine (Experimental Example 7). It is a drawing substitute graph which shows the result of Experimental example 7 shown in FIG. It is a figure which shows the process of the passive avoidance task (Passive avoidance test) used for examination (Experiment 8) of the relationship between forgetting hippocampus-dependent fear memory and memantine. 16 is a drawing substitute graph showing the results of Experimental Example 8 shown in FIG. 15. It is a figure which shows the process of the fear condition context task (Contextual Conditioning Test) used in order to show the relationship (experimental example 9) of the process of promoting neurogenesis in a hippocampus, and memory forgetting. It is a drawing substitute graph which shows the result of the experiment example 9 of FIG.

  As will be described later in detail in the Examples, the present inventor recalled horror memory to the extent of inducing gene expression in the hippocampus for non-human animals subjected to a fear-conditioning context learning task by using a mouse. After that, it was newly found that the old fear memory that lost hippocampal dependence was forgotten for a long time after storage by subjecting the non-human animal to the step of promoting neurogenesis in the hippocampus.

  In this regard, the present invention is based on the concept that traumatic trauma (trauma) revives the hippocampal dependence of fear memory, and then promotes neurogenesis in the hippocampus, so that the new neurons control the fear memory circuit. As a result, it is thought that forgetting the fear memory encoded in the memory circuit was promoted.

Therefore, the present invention can be suitably used for applying to a patient with post-traumatic stress disorder (PTSD) and treating the post-traumatic stress disorder (PTSD).
It can also be used to shorten the treatment time of continuous exposure therapy (PE), which is a treatment for post-traumatic stress disorder (PTSD).

<Screening method for therapeutic agents for post-traumatic stress disorder (PTSD) using non-human animals subjected to fear conditioning contextual learning tasks>
The formation of fear memory is one of the instinctive behaviors of all living things, from insects to higher organisms. For this reason, the Contextual Fear Conditioning Test is considered an optimal model for clarifying the memory formation mechanism. In this Contextual Fear Conditioning Test, if an increase in the re-fixation of fear-conditioned memory is seen, it is believed to be associated with post-traumatic stress disorder (PTSD).

  The screening method of the present invention utilizes the fact that neurogenesis in the hippocampus prompts forgetting fear memory. Therefore, a test substance that can promote neurogenesis in the hippocampus is administered to a non-human animal, and the influence of this test substance on the animal is detected by using forgetting of old memory in the animal as an index. For the treatment of post-traumatic stress disorder (PTSD).

  Any substance can be used as the test substance, and the type of the test substance is not particularly limited, and may be, for example, a peptide, protein, or synthetic compound, and further present in a natural extract. Plant extracts, animal tissue extracts, microbial extracts, food ingredients, and the like. The test substance may be a novel compound or a known compound. Furthermore, as this test substance, memantine, which is a therapeutic agent that has been conventionally effective for memory impairment, for example, is known as a therapeutic agent for Alzheimer-type dementia. Examples of the test substance include antidepressants that promote neurogenesis in the hippocampus. If such a conventionally used therapeutic agent for each disease type can be assayed as a therapeutic agent for post-traumatic stress disorder (PTSD), a therapeutic agent with high safety and practicality can be provided.

  Examples of the method for administering a test substance to a non-human animal include intraperitoneal administration and intravenous injection, and may be appropriately selected in view of the type of non-human animal, the nature of the test substance, and the like. The dose of the test substance is determined in consideration of the type of dosage form, the administration method, the age and weight of the administration subject (including animals), the symptoms, and the like.

  The “forgetting” of memory in the present invention is the ability of a non-human animal to forget things, and refers to a state in which long-term memory accumulated in the brain is lost and cannot be recalled.

  In the present invention, the non-human animal is a non-human animal, preferably a mammal, and examples thereof include mice, rats, guinea pigs, hamsters, rabbits, goats and pigs, more preferably rodents. It is an animal, more preferably a mouse.

  The “fear-conditioning context learning task” in the present invention uses an object (context) that is sensed by the five senses such as vision, hearing, and smell as a conditioned stimulus (Conditioned Stimulus; CS), while an electric shock or the like is an unconditioned stimulus (Unconditioned Stimulus; US). In the present invention, in order to promote the formation of hippocampus-dependent memory, the conditional stimulus (CS) is more preferably a place such as a chamber.

  In the first step of the present invention, “the state in which the fear memory is converted into the hippocampus-independent memory” means that a certain period of time has elapsed since the fear memory is formed in the brain, and the fear memory is the hippocampus. It is a state recalled depending on the cerebral cortex. Specifically, it means a state where 4 to 8 weeks have passed since the non-human animal was subjected to the fear conditioning contextual learning task, and more preferably 8 weeks.

  In addition, in the first step of the present invention, the time for returning to the chamber used for the fear conditioning context learning task to recall the fear memory is 3 minutes from the inventor's knowledge, and even 5 minutes is insufficient. (Non-Patent Document 1), preferably at least 10 minutes.

  As described above, “re-immobilization” of fear memory requires expression of a gene group typified by an early response gene in the hippocampus. For this reason, in the first step of the present invention, “inducing gene expression in the hippocampus” indicates a state in which the hippocampus is activated by recalling fear memory, and the hippocampal dependence of fear memory is restored. Shows the state in which memory is guided to the process of refixation.

  In the present invention, the “gene” is not particularly limited, but is preferably an early response gene whose expression is rapidly and transiently induced in brain neurons, for example, c-fos or Egr− There are a gene encoding a transcriptional regulatory factor such as 1 and a gene encoding a protein related to a synapse such as Arc, and the c-fos gene is more preferable.

<1. Study on the relationship between hippocampus-dependent fear memory oblivion and memantine>
As described above, in the case of fear memory that is recalled depending on the hippocampus, the present inventor returns the mouse to the same chamber as the training for 3 minutes to recall the fear memory. The destruction of the memory was observed by intraperitoneal administration of mycin (ANI). In addition, it has been clarified that administration of the memantine enhances neurogenesis in the hippocampus, and that young neurons that have just matured contribute to the improvement of memory formation ability. On the other hand, there is a view that newly produced neurons are incorporated into the neural circuit when mature, and this may forget the memory accumulated in the neural circuit (Meltzer etal., Trends in Neuroscience). , 2005; Weisz & Argibay, Cognition, 2012). From these points, the present inventor examined the relationship between the memantine and forgetting hippocampus-dependent fear memory. In addition, based on the knowledge of the present inventor (Non-patent Document 3), for the forgotten hippocampus-dependent fear memory when memantine is continuously administered in order to continuously produce mature young neurons. Also examined.

  This experimental example 1 was performed using a Contextual Fear Conditioning Test. FIG. 1 shows this Contextual Fear Conditioning Test. The formation of fear memory is one of the instinctive behaviors of all living things, from insects to higher organisms. For this reason, the Contextual Fear Conditioning Test is considered an optimal model for clarifying the memory formation mechanism.

  In this Experimental Example 1, C57BL / 6N strain mice (8 weeks of age or older) were used as animals. This mouse was purchased from Oriental Yeast Co., Ltd. As an experimental group of Experimental Example 1, a mouse group (hereinafter referred to as “memantine continuous administration group”) in which memantine (manufactured by Sigma Aldrich Japan GK) was intraperitoneally administered once a week for a total of four times was used. . In addition, a group of mice in which memantine was administered intraperitoneally only once (hereinafter referred to as “memantine single administration group”) was used. As a control group, a group of mice to which a solvent (Saline) was intraperitoneally administered was used.

As the Contextual Fear Conditioning Test, the mouse was first placed in a chamber (manufactured by Ohara Medical Sangyo Co., Ltd.) with wires on the floor for training (fear conditioning) for 3 minutes, and then 148 After a second, a 0.4 mA electric shock was applied for 2 seconds, and each mouse was trained to associate a fear experience (electric shock) with a feared environment (context). Twenty-four hours later, as test 1, the mice in each group were again returned to the same chamber as the training for 5 minutes to recall the fear memory (re-exposure). At this time, the ratio of freezing time exhibited by the mouse was measured, and this ratio was used as an index of fear memory. It can be evaluated that a stronger fear memory was formed in the mouse as the ratio of the freezing time of the mouse was longer.

  Furthermore, in the memantine continuous administration group, after test 1, memantine was administered once a week for a total of four times (4 weeks). Thereafter, the mouse was returned to the same chamber as the training for 5 minutes, and the percentage of the mouse's freezing reaction time was measured (Test 2). On the other hand, in the memantine single administration group, memantine was administered only once within one week after test 1. Three weeks later, that is, at the same time as the memantine continuous administration group, the mice were returned to the same chamber as the training for 5 minutes, and the percentage of the mice's freezing reaction time was measured (test 2). For the control group, the solvent (Saline) was administered only once within one week after test 1. Three weeks later, that is, at the same time as the memantine continuous administration group, the mice were returned to the same chamber as the training for 5 minutes, and the percentage of the mice's freezing reaction time was measured (test 2). Here, the single dose of the memantine is 50 mg / kg body weight. In the control group, the same volume of solvent (Saline) as memantine was administered.

  In Experimental Example 1, since test 1 was performed 24 hours after the formation of fear memory for each group of mice, the percentage of freezing time indicated by each group was determined in the hippocampus. It can be said that this is due to the fear memory recalled depending on the patient (hereinafter also referred to as “hippocampus-dependent fear memory”).

  The results are shown in FIG. 2, (A) shows the percentage of freezing reaction time shown by the control group during training, (B) shows the percentage of freezing reaction time shown by the single administration group of memantine during training, (C) Indicates the percentage of freezing reaction time indicated by the memantine continuous administration group during training. In FIG. 2, (D) shows the percentage of freezing reaction time shown by the control group at the time of test 1, and (E) shows the percentage of freezing reaction time shown by the single administration group of memantine at the time of test 1. , (F) shows the percentage of freezing reaction time shown by the memantine continuous administration group at the time of test 1. In FIG. 2, (G) shows the percentage of freezing reaction time shown by the control group at the time of test 2, and (H) shows the percentage of freezing reaction time shown by the single administration group of memantine at the time of test 2. , (I) shows the percentage of freezing reaction time shown by the memantine continuous administration group at test 2.

  In this Experimental Example 1, 10 mice were used for each group, and the results are shown as mean value ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  As shown in FIG. 2, the percentage of freezing reaction time during test 1, that is, recalling fear memory, was not significantly different among all groups, indicating that fear memory was formed after training, re-exposure It was confirmed that fear memory was recalled. And in Test 2, the memantine single administration group (H) has a lower rate of freezing reaction time than the control group (G), suggesting that fear memory can be forgotten by administration of memantine (MEM). It was done. Further, in the memantine continuous administration group (I), the proportion of freezing reaction time is lower than in the memantine single administration group (H), and a significant difference is observed with respect to the control group (G). It was more prominently suggested that the fear memory can be forgotten by administration.

  From the results of Experimental Example 1, it is clear that hippocampal-dependent fear memory can be forgotten by administering memantine in a period when hippocampus-dependent fear memory is formed and hippocampus dependence is maintained. It became. Furthermore, it has been clarified that continuous administration of memantine induces hippocampal-dependent fear memory forgetting.

<2. Examination of the relationship between oblivion of old memory and memantine>
The results of Experimental Example 1 revealed that hippocampus-dependent fear memory was forgotten due to the administration of the memantine. Therefore, the administration of the memantine was not dependent on the hippocampus but depended on the cerebral cortex. The relevance of forgotten memories (hereinafter referred to as “old memories”) was investigated.

  Similar to Experimental Example 1, this examination was performed using a fear conditional context task (Contextual Fear Conditioning Test). FIG. 3 shows this Contextual Fear Conditioning Test. In Experimental Example 2, C57BL / 6N strain mice (8 weeks of age or older) were used as animals. This mouse was purchased from Oriental Yeast Co., Ltd. As the experimental group of Experimental Example 2, a mouse group in which memantine was administered intraperitoneally (hereinafter referred to as “memantine-administered group”) was used. As this control, a group of mice to which a solvent (Saline) was intraperitoneally administered was used (hereinafter referred to as “control group”).

  In Experimental Example 2, first, training (fear conditioning) was performed by the same method as in Experimental Example 1. In Example 2, no re-exposure was performed, and after 8 weeks from the training, memantine or a solvent (Saline) was intraperitoneally administered once a week. Memantine or vehicle (Saline) was administered four times (4 weeks) in total. The single dose of memantine was 50 mg / kg body weight. The same volume of solvent (Saline) was administered to the control group.

  Finally, after the end of memantine or vehicle (Saline) administration (12 weeks after training), each mouse was returned to the same chamber as the training again for 5 minutes as a test, and the percentage of freezing time indicated by the mice Was measured.

  In Experimental Example 2, memantine or a solvent (Saline) was administered from 8 weeks after learning fear memory for each group of mice, and then the test was performed. The rate of freezing time can be attributed to fear memory recalled not by the hippocampus but by the cerebral cortex. In this Experimental Example 2, 10 mice were used in each group, and the results were shown as mean value ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  The results are shown in FIG. In FIG. 4, (A) shows the percentage of freezing reaction time shown by the control group during training, and (B) shows the percentage of freezing reaction time shown by the memantine administration group during training. In FIG. 4, (C) shows the percentage of freezing reaction time shown by the control group during the test, and (D) shows the percentage of freezing reaction time shown by the memantine administration group during the test.

  As shown in FIG. 4, the ratio of the freezing reaction time during the test shown by the memantine administration group (D) was comparable to that of the control group (C). From this result, it became clear that even when memantine was administered to promote neurogenesis in the hippocampus, “hippocampal-independent” old fear memories were not forgotten.

<3. Examination of novel gene induction in re-immobilization of old fear memory>
It has been clarified by the present inventor that reimmobilization of hippocampus-dependent fear memory requires the expression of early response genes in the hippocampus, that is, activation of the hippocampus (Suzuki, A., Mukawa, T., Tsukagoshi, A., Frankland, PW & * Kida, S. Activation of LVGCCs and CB1 receptors required for destabilization of reactivated contextual fear memories. Learn. Mem. 15, 426-33,2008, Mamiya , N., Fukushima, H., Suzuki, A., Matsuyama, Z., Homma, S., Frankland, PW & * Kida, S. Brain region-specific gene expression activation required for reconsolidation and extinction of contextual fear memory. ).

  Further, from the results of Experimental Example 1, hippocampus-dependent fear memory was formed, and memantine was administered at the time when hippocampus dependence was maintained, that is, by promoting neurogenesis in the hippocampus, hippocampus-dependent It became clear that fear memories could be forgotten. Based on these findings, the present inventor considered that even if the memory is old, it is forgotten by promoting early gene expression in the hippocampus, ie, restoring hippocampal dependence. Therefore, we hypothesized that hippocampal dependence would be restored by inducing re-fixation of old memory.

Here, the transcriptional regulatory factor CREB (cAMP-responsive element binding protein) existing downstream of the Ca ²⁺ and cAMP signaling pathways contributes to the re-immobilization of memory. The c-fos gene, which is a target gene of this transcriptional regulatory factor CREB and is a marker of neural activity, exists.
In this Experimental Example 3, whether or not gene expression by the transcriptional regulatory factor CREB is induced in the hippocampus using the immunohistochemical staining and recalling the old memory, using the expression level of the c-fos gene as an index in the hippocampus. Whether or not was activated was examined. Note that the animals and experimental instruments used in this Experimental Example 3 are the same as in Experimental Example 1.

FIG. 5 shows an experimental procedure of the third experimental example. In this Experimental Example 3, as an experimental group, a mouse was placed in a chamber (conditional stimulus: CS) in which wires were placed on the floor for 3 minutes, and after 148 seconds, 0.4 mA electric shock was given for 2 seconds for training ( Fear conditioning). Four weeks after this training, a test (re-exposure) was used that again brought the mouse back into the same chamber as the training for 10 minutes and recalled fear memory.
As a first control group as a control, mice were placed in a chamber (conditional stimulus: CS) in which wires were placed on the floor for 3 minutes, and training was performed without applying an electric shock as an unconditional stimulus (US). Four weeks after this training, a test in which the mice were returned to the same chamber as the training again for 3 minutes was used as a test.
As a second control group, mice were placed for 3 minutes in a chamber (conditional stimulus: CS) in which electric wires were placed on the floor surface, and training was performed without applying an electric shock as an unconditional stimulus (US). Four weeks after this training, a test in which the mice were returned to the same chamber as the training again for 10 minutes was used as a test.
As a third control group, the mouse was placed in a chamber (conditional stimulus: CS) with electric wires on the floor for 3 minutes, and after 148 seconds, 0.4 mA electric shock was given for 2 seconds for training (fear conditioning) Went. Four weeks after this training, as a test (re-exposure), the mouse was returned to the same chamber as that for the training again for 3 minutes and used to recall fear memory.

  Furthermore, in each group of mice, perfusion fixation (Per. Fix.) Was performed 90 minutes after the test, and then immunohistochemical staining was performed on a section containing the hippocampal region to be detected. The perfusion fixation and immunohistochemical staining were performed by known methods, and an antibody that recognizes the c-fos gene product (protein) was used in the immunohistochemical staining.

  The number of c-fos positive cells present in a 100 μm square within the specific regions (CA1 region and CA3 region of the hippocampus) of the section subjected to the immunohistochemical staining was counted. This work was performed at three locations for each brain region. In this Experimental Example 3, 10 mice were used in each group, and the results are shown as mean value ± standard error. Comparison of each data was statistically analyzed using analysis of variance. When p <0.05, it was judged that there was a significant difference.

  The results are shown in FIG. 6 (a) shows the number of c-fos positive cells in the CA1 region of the hippocampus, and FIG. 6 (b) shows the number of c-fos positive cells in the CA3 region of the hippocampus. In FIG. 6, (A) shows the number of c-fos positive cells in the first control group, (B) shows the number of c-fos positive cells in the second control group, and (C) shows c-fos in the third control group. (D) shows the number of c-fos positive cells in the experimental group.

  As shown in FIG. 6, in the CA1 region of the hippocampus, the number of c-fos positive cells in the experimental group (D) is c- in the other control groups (A) to (C), particularly the third control group (C). The value was significantly higher than the number of fos positive cells.

  Also in the CA3 region of the hippocampus, the number of c-fos positive cells in the experimental group (D) is the number of c-fos positive cells in the other control groups (A) to (C), particularly the third control group (C). The value was significantly higher than.

In this Experimental Example 3, since the test is performed four weeks after the training, the fear memory is generally a memory that does not depend on the hippocampus. However, given that the number of cells expressing c-fos gene in the hippocampus is high, the results of this Experimental Example 3 indicate that the hippocampus is activated when recalled for a long time even in old memory (genes in the hippocampus). It was revealed that expression is induced).
Furthermore, this suggests that re-immobilization of old memory requires gene expression in the hippocampus.
In addition, since the number of c-fos positive cells in the experimental group (D) is higher than that in the third control group (C) in which the time for recalling fear memory is 3 minutes, However, it is clear that recalling for a short period of time is not enough, but recalling for a long period of time makes it dependent on the hippocampus. In other words, it is possible to determine that hippocampal dependence has been restored by recalling old memories that are not hippocampus dependent for a long time.

<4. Examination of the need for novel gene expression in the re-fixation of old fear memories>
Based on the results of Experimental Example 3, it was thought that hippocampal dependence was restored by recalling for a long time even if it was an old memory. Therefore, using the local injection method of anisomycin (ANI), a protein synthesis inhibitor, The effect of gene expression inhibition in the hippocampus was examined.

  FIG. 7 shows an experimental procedure of the fourth experimental example. In this Experimental Example 4, as the experimental group, a group of mice in which fear memory was recalled by returning to the chamber for 10 minutes (re-exposure) and anisomycin (ANI) was locally injected into the hippocampus using a cannula was used. As the first control group as a control, a group of mice in which fear memory was recalled by returning to the chamber for 3 minutes (re-exposure) and a solvent (Saline) was locally injected into the hippocampus using a cannula was used. As a second control group, a group of mice which were returned to the chamber for 3 minutes to recall fear memory (re-exposure) and anisomycin (ANI) was locally injected into the hippocampus using a cannula was used. Furthermore, as a third control group, a group of mice in which fear memory was recalled by returning to the chamber for 10 minutes (re-exposure) and a solvent (Saline) was locally injected into the hippocampus using a cannula was used. The dose of anisomycin (ANI) was 62 μg (total amount 0.5 μl) / side. The same volume of solvent (Saline) was administered to each control group.

  In Experimental Example 4, training (fear conditioning) was performed on the mice in each group by the same method as in Experimental Example 1. Four weeks after this training, Test 1 (re-exposure) was performed to remind mice of fear memory. This test 1 (re-exposure) recalled fear memory by returning the mouse to the same chamber as the training for 10 minutes as described above in the experimental group. In the first control group and the second control group, as described above, the mice were returned to the same chamber as the training for 3 minutes to remind them of fear memory. In the third control group, as described above, the mice were returned to the same chamber as the training for 10 minutes to recall fear memory. Then, as test 2, 24 hours after test 1, each group of mice was returned again to the same chamber as the training for 5 minutes, and the percentage of freezing time of the mice was measured. The anisomycin (ANI) or solvent (Saline) was locally injected immediately after the mouse was removed from the chamber.

  The results are shown in FIG. In FIG. 8A, (A) shows the percentage of freezing reaction time shown by the first control group at the time of test 1, and (B) shows the percentage of freezing reaction time shown by the second control group at the time of test 1. Indicates. (C) shows the percentage of freezing reaction time shown by the first control group at the time of test 2, and (D) shows the percentage of freezing reaction time shown by the second control group at the time of test 2. On the other hand, in FIG. 8B, (E) shows the percentage of freezing reaction time shown by the third control group at the time of test 1, and (F) shows the percentage of freezing reaction time shown by the experiment group at the time of test 1. Indicates. (G) shows the percentage of freezing reaction time shown by the third control group at the time of test 2, and (H) shows the percentage of freezing reaction time shown by the experimental group at the time of test 2. In this Experimental Example 4, 8 mice were used for the first control group and the second control group, and 11 mice were used for the third control group and the experimental group, and the results are shown as mean value ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  As shown in FIG. 8, in Test 2, the ratio of freezing reaction time shown by the experimental group (H) was significantly lower than the freezing reaction time ratio shown by the third control group (G). Further, the ratio of the freezing reaction time shown by the experimental group (H) in Test 2 was significantly lower than the freezing reaction time ratio shown by the experimental group (F) in Test 1. This is consistent with the result that gene expression was induced when old memory was recalled for a long time (Experimental Example 3), and reimmobilization of old fear memory by gene expression inhibition by administration of anisomycin (ANI). Is thought to have been disturbed (the fear memory was destroyed).

  According to the results of the present experimental example 4 and the experimental example 3, even if it is an old fear memory, by recollecting for a long time, (i) hippocampal dependence is restored, (ii) fear memory is re-stored Therefore, it has become clear that re-immobilization that requires new gene expression in the hippocampus is required.

<5. Examination of hippocampal dependence revival related to old memories recalled for a long time>
Based on the results of Experimental Example 3, it was thought that hippocampal dependence would be restored by recollecting old memory for long periods of time. Therefore, lidocaine (Lido), a neuronal activity inhibitor, was used for a long time. After recalling, we examined whether the hippocampal dependence of the memory was restored.

FIG. 9 shows the experimental procedure of this Experimental Example 5. FIG. 10 shows the results of this experimental example 5. In this Experimental Example 5, as the experimental group, a group of mice in which the fear memory was recalled by returning to the chamber for 10 minutes (re-exposure) and lidocaine (Lido) was locally injected into the hippocampus using a cannula 24 hours later was used. (B, D, F, H in FIG. 10).
As a control group to be used as a control, a group of mice which were brought back to the chamber for 10 minutes to recall fear memory (re-exposure), and 24 hours later, a solvent (VEH) was locally injected into the hippocampus using a cannula was used ( A, C, E, G in FIG. The dose of lidocaine (Lido) was 62 μg (total amount 0.5 μl) / side. Each control group received the same volume of solvent (VEH).

In Experimental Example 5, training (fear conditioning) was performed on the mice of each group by the same method as in Experimental Example 1. Four weeks after this training, Test 1 (re-exposure) was performed on mice in each group, which recalled fear memory. In Test 1 (re-exposure), as described above, each group of mice was returned to the same chamber as the training for 10 minutes to recall fear memory.
Then, as test 2, 24 hours after test 1, each group of mice was returned again to the same chamber as the training for 5 minutes, and the percentage of freezing time of the mice was measured. Lidocaine (Lido) or vehicle (VEH) was locally injected 10 minutes before each group of mice was returned to the chamber.
Furthermore, as test 3, 24 hours after test 2, each group of mice was returned to the same chamber as the training for 5 minutes again, and the percentage of freezing time of the mice was measured. Prior to Test 3, no lidocaine (Lido) or solvent (VEH) was locally injected in either group.

The results are shown in FIG. In FIG. 10, (A) shows the percentage of freezing reaction time shown by the control group during training, and (B) shows the percentage of freezing reaction time shown by the experimental group during training. (C) shows the percentage of freezing reaction time shown by the control group at the time of test 1, and (D) shows the percentage of freezing reaction time shown by the experimental group at the time of test 1. Furthermore, (E) shows the percentage of freezing reaction time shown by the control group at the time of test 2, and (F) shows the percentage of freezing reaction time shown by the experimental group at the time of test 2. (G) shows the percentage of freezing reaction time shown by the control group at the time of test 3, and (H) shows the percentage of freezing reaction time shown by the experimental group at the time of test 3.
In this Experimental Example 5, 11 mice were used for the experimental group and the control group, and the results are shown as mean value ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

As shown in FIG. 10, in Test 2 performed after the hippocampus was inactivated using lidocaine (Lido), a neuronal activity inhibitor, the percentage of freezing reaction time shown by the experimental group (F) Compared with the ratio of the freezing reaction time shown by the experimental group (D) in FIG.
Moreover, the ratio of the freezing reaction time shown by the experimental group (F) in Test 2 was significantly lower than the freezing reaction time ratio shown by the control group (E) in Test 2.
Furthermore, the percentage of freezing reaction time shown by the experimental group (F) in Test 2 is the freezing time indicated by the experimental group (H) in Test 3 which was conducted without locally injecting lidocaine (Lido), a neuronal activity inhibitor. The value was significantly lower than the reaction time ratio.
These are consistent with the results of experimental gene expression when recalling old memory for a long time (Experimental Example 3) and the results of memory disruption by inhibiting gene expression in the hippocampus (Experimental Example 4), Since hippocampal dependence of memory was restored by long-term recall, it is considered that memory was not recalled by hippocampal inactivation by administration of lidocaine (Lido).

In addition to the results of Experimental Example 3 and Experimental Example 4, according to the result of Experimental Example 5, even if it is an old fear memory, (i) hippocampal dependence is restored by recalling for a long time. It became clear.
In addition, in this Experimental Example 5, lidocaine (Lido) is used as a neuron activity inhibitor. However, this neuron activity inhibitor is not particularly limited as long as it can inactivate the hippocampus. These known inhibitors may be used. In such a case, for example, CNQX (6-cyano-7-nitroquinoxalinedione-2,3-dione), which is a non-NMDA type (AMPA type / kainic acid type) receptor antagonist, can also be used.

<6. Examination of the relationship between oblivion of old memory and memantine>
From Experimental Example 1, it was clarified that if the memory is a hippocampus-dependent fear memory, the fear memory is forgotten by administering memantine within a period in which the fear memory shows hippocampal dependence. On the other hand, according to the result of Experimental Example 2, in the case of old fear memory, even when memantine contributing to memory forgetting was administered, forgetting of the fear memory was not observed. From the results of Experimental Example 3 and Experimental Example 4, the present inventor has clarified that the hippocampal dependence of the memory is restored by recalling the old memory for a long time (10 minutes). Based on these facts, the present inventor considered that even if it was an old fear memory, the memory was forgotten by administering memantine after recalling this old fear memory for a long time.

  Similar to Experimental Example 1, Experimental Example 6 was conducted using a Contextual Fear Conditioning Test. FIG. 11 shows this fear conditional context test (Contextual Fear Conditioning Test). The same animal and chamber as those used in Experimental Example 1 were used. Further, the dosage of memantine and a solvent (Saline) described later is the same as in Experimental Example 1. The number of administrations of memantine and the solvent (Saline) was set to once a week for a total of 4 times (4 weeks) from the results of Experimental Example 2.

In this Experimental Example 6, as an experimental group, a time period for returning to the chamber in test 1 (re-exposure) after training was 3 minutes, and then a mouse group administered memantine (hereinafter referred to as “first memantine-administered group”); A group of mice (hereinafter referred to as “second memantine administration group”) in which the time for returning to the chamber was 10 minutes and then memantine was administered was used. In addition, as a control group, a test group 1 (re-exposure) after training had a period of 3 minutes for returning to the chamber, and thereafter a group of mice (hereinafter referred to as “first control group”) administered with a solvent (Saline); A group of mice (hereinafter referred to as “second control group”) in which the return time was 10 minutes and then the solvent (Saline) was administered was used.

  Training (fear conditioning) was performed in the same manner as in Experimental Example 1. After 8 weeks from this training, mice in each group were subjected to the re-exposure as test 1 and the percentage of freezing time exhibited by the mice was measured. Furthermore, as described above, memantine and vehicle (Saline) were administered four times (4 weeks), and then as test 2, the mice in each group were returned again to the same chamber as the training for 5 minutes, and the freezing reaction time indicated by the mice The ratio of (Freezing Time) was measured.

The results are shown in FIG. Each symbol shown in FIG. 12 represents the following values, (A) to (D) are ratios of freezing time indicated by each group during training, and (E) to (H) are tests. The percentage of freezing time shown by each group at 1 (re-exposure), (I) to (L) are the percentage of freezing time shown by each group at test 2 Indicates.
(A) Proportion of freezing time indicated by the first control group (B) Proportion of freezing time indicated by the second control group (C) Freezing time indicated by the first memantine administration group Proportion of freezing time (D) Proportion of freezing time shown by second memantine administration group (E) Proportion of freezing time shown by first control group (F) Proportion of freezing time indicated by the second control group (G) Proportion of freezing time indicated by the first memantine administration group (H) Freezing time indicated by the second memantine administration group Ratio of (Freezing Time) (I) Ratio of freezing time shown by the first control group (J) Ratio of freezing time shown by the second control group (K) Proportion of freezing time indicated by the mantin administration group (L) Proportion of freezing time indicated by the second memantine administration group

As shown in FIG. 12, the percentage of freezing reaction time at test 1 (re-exposure) is not significantly different among all groups, and fear memory is formed after training, and fear memory is recalled by re-exposure. It has been confirmed. In addition, the ratio of freezing time shown by the first memantine administration group at the time of Test 2 was the same value as that of the first control group and the second control group. On the other hand, the ratio of freezing time shown by the second memantine administration group at the time of test 2 was significantly lower than that of the other groups.
In this Experimental Example 6, 10 mice were used for the first control group and the first memantine administration group, 12 mice were used for the second control group, and 13 mice were used for the second memantine administration group. It showed in. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  From the results of this experimental example 6, with regard to old memory, simply recalling fear memory by returning to the chamber for 3 minutes does not forget the fear memory even if memantine is administered continuously for 4 weeks. It was revealed that they were forgotten by recalling long-term fear memories (i) reviving hippocampal dependence, and (ii) subsequent administration of memantine for 4 consecutive weeks.

<7. Examination of relationship between memantine and induction of re-fixation of hippocampus-dependent fear memory>
According to Experimental Example 6, it has been clarified that administration of memantine for promoting neurogenesis forgets the old memory in which hippocampal dependence has been restored,
We examined whether re-immobilization induction after long-term recall is necessary for forgetting old fear memory by the memantine.

  Here, the present inventor is immobilized due to the administration of a calcineurin activity inhibitor, the memory held is prevented from being restored to an unstable state by recall, and as a result, re-immobilized (Hotaka Fukushima, Yue Zhang, Georgia Archbold, Rie Ishikawa, Karim Nader, Satoshi Kida “Enhancement of fear memory by retrieval through reconsolidation” The elifesciences June 24, 2014)

For this reason, in this Experimental Example 7, it is inhibited that the old memory is returned to an unstable state by intraperitoneally administering a calcineurin activity inhibitor to the mouse 5 minutes before the test 1 (re-exposure) after training. (See FIG. 13), and examined whether reimmobilization induction after long-term recall is necessary for forgetting old fear memory by the memantine.
The experimental procedure of Experimental Example 7 is mainly the same as the experimental procedure of Experimental Example 6, and the calcineurin activity inhibitor was intraperitoneally administered to mice 5 minutes before Test 1 (re-exposure) after training. It differs from Experimental Example 6 in that the step of inhibiting the old memory from being returned to an unstable state is performed, and the time for returning to the chamber in Test 1 is 10 minutes in all groups of mice (FIG. 6). 13). For this reason, in the following description, the description of the same process is omitted.

In this experimental example 7, as an experimental group, FK506, a calcineurin activity inhibitor, was administered intraperitoneally 5 minutes before test 1 (re-exposure) after training, and the time for returning to the chamber in test 1 was 10 minutes. Thereafter, a group of mice administered with memantine was used.
In addition, as a control group, a group of mice (hereinafter referred to as “Sine”) in which the solvent (VEH) was intraperitoneally administered 5 minutes before Test 1 and the time for returning to the chamber in Test 1 was 10 minutes, and then the solvent (Saline) was administered. “First control group”) and FK506, which is a calcineurin activity inhibitor, was intraperitoneally administered 5 minutes before Test 1, and the time for returning to the chamber in Test 1 was 10 minutes, followed by administration of a solvent (Saline). Mice (hereinafter referred to as “second control group”) and the solvent (VEH) was intraperitoneally administered 5 minutes before Test 1, and the time for returning to the chamber in Test 1 was 10 minutes, followed by administration of memantine. Mouse group (hereinafter, “third control group”) was used.

The results are shown in FIG. Each symbol shown in FIG. 14 represents the following values, (A) to (D) are ratios of freezing time indicated by each group during training, and (E) to (H) are tests. The percentage of freezing time shown by each group at 1 (re-exposure), (I) to (L) are the percentage of freezing time shown by each group at test 2 Indicates.
(A) Proportion of freezing time indicated by the first control group (B) Proportion of freezing time indicated by the second control group (C) Freezing time indicated by the third control group Proportion of freezing time (D) Proportion of freezing time indicated by experimental group (E) Proportion of freezing time indicated by first control group (F) Second control group (G) Proportion of freezing time indicated by the third control group (H) Proportion of freezing time indicated by the experimental group (I) ) Proportion of freezing time indicated by the first control group (J) Proportion of freezing time indicated by the second control group (K) Freezing time indicated by the third control group Freezing time ratio (L) Freezing time ratio shown by the experimental group

As shown in FIG. 14, the ratio of freezing time at test 1 (re-exposure) was not significantly different among all groups, and that fear memory was formed after training, and re-exposure It was confirmed that fear memory was recalled.
In addition, the ratio of freezing time shown by the experimental group at the time of test 2 showed a value similar to that of the first control group and the second control group. On the other hand, the ratio of freezing time shown by the third control group at the time of test 2 was significantly lower than that of the other groups.
In this Experimental Example 7, 10 mice were used in all groups, and the results were shown as mean ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

From the results of Experimental Example 7, it was revealed that memantine returned to the chamber for 10 minutes and reminded of fear memory for a long time to forget only the old memory in which hippocampal-dependent re-fixation was induced. In other words, it was confirmed that the hippocampal dependence revived with the re-immobilization induction in the hippocampus.
In this Experimental Example 7, FK506 is used as a calcineurin activity inhibitor. However, the calcineurin activity inhibitor is not particularly limited, and can inhibit memory destabilization after recall. Other known inhibitors may be used. In such a case, for example, cycloporin A can be used.

<8. Examination of the relationship between hippocampus-dependent fear memory oblivion and memantine>
According to the result of Experimental Example 6, with regard to old memory, simply returning to the chamber for 3 minutes and reminiscent of fear memory does not forget the fear memory even if memantine is administered continuously for 4 weeks, and returns to the chamber for 10 minutes. It was revealed that, by recalling fear memory for a long time, (i) reviving hippocampal dependence, and (ii) subsequent administration of memantine continuously for 4 weeks, it was forgotten.
Based on these results, we investigated the relationship between hippocampus-dependent fear memory and memantine using a passive avoidance test, a learning task that is said to be hippocampus-dependent.

  In Experimental Example 8, a group of mice to which memantine was administered after training was used as the experimental group. As a control group, a group of mice administered with a solvent (Saline) after training was used. Here, the dosages of memantine and solvent (Saline) are the same as in Experimental Example 1.

In this Experimental Example 8, as shown in FIG. 15, as a training, each group of mice is placed in a light box of a device in which a light box and a dark box are connected, and a mouse that dislikes the light box enters the dark box 0.1 seconds later. A shock of mA was given. At this time, the time to enter the dark box (hereinafter referred to as “crossover latency”) was measured.
For the mice in the experimental group, memantine was administered 24 hours after the training, and then memantine was administered four times in total (4 weeks) every week thereafter.
For the mice in the control group, the solvent (Saline) was administered 24 hours after the training, and thereafter, a total of four times (4 weeks) of the solvent (Saline) was administered every week thereafter.
One week after the administration of these memantine and the solvent (Saline), as a test, each group of mice was again placed in a light box of the same apparatus.
In the test, the crossover latency was measured as in the training.

  The results are shown in FIG. In FIG. 16, (A) shows the crossover latency of the control group during training, and (B) shows the crossover latency of the experimental group during training. (C) shows the crossover latency of the control group during the test, and (D) shows the crossover latency of the experimental group during the test.

As shown in FIG. 16, the crossover latency of the experimental group at the time of the test was significantly lower than the crossover latency of the control group at the time of the test.
In this Experimental Example 8, 11 mice were used for the experimental group and 10 mice were used for the control group, and the results are shown as mean value ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  From the results of Experimental Example 8, it was clarified that memantine was forgotten even in a hippocampus-dependent fear memory of a type different from fear conditioning memory by continuously administering memantine for 4 weeks. Therefore, in combination with the results of Experimental Example 6, it was confirmed that memantine administration for 4 weeks leads to hippocampal-dependent fear memory forgetting.

<9. Confirmation of the relationship between the process of promoting neurogenesis in the hippocampus and memory forgetting>
As mentioned above, memantine enhances neurogenesis in the hippocampus. Then, according to the result of Experimental Example 6, return to the chamber for 10 minutes, recall a long time fear memory (i) restore hippocampal dependence, and (ii) then administer memantine continuously for 4 weeks It became clear that fear memories were forgotten. Here, as a thing that enhances neurogenesis in the hippocampus, the “exercising task (Running)” is known (Nat Neurosci. 1999 Mar; 2 (3): 266-70.Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.van Praag H1, Kempermann G, Gage FH.). For this reason, the present inventor considers that the memory is forgotten by stimulating neurogenesis in the hippocampus using a motor task after reviving hippocampal dependence by recalling old fear memory for a long time Went.

In this Experimental Example 9, the animals used, the training (fear conditioning) method, the test 1 (re-exposure) method, the test 2 method, the period from training to test 1 and the period from test 1 to test 2 are: This is the same as that of Experimental Example 6. On the other hand, in the present experimental example 9, the method from test 1 (re-exposure) to test 2 is different (see FIG. 17).
That is, in Experimental Example 8, using a running wheel (manufactured by Marcan Co., Ltd.) as a means for promoting neurogenesis in the hippocampus, the neurogenesis in the hippocampus was promoted by exercise.

In Experimental Example 9, the following groups of mice were used.
(1) In test 1 (re-exposure) after training, the group was returned to the chamber for 10 minutes and then used for exercise tasks (hereinafter referred to as “running group”).
(2) A group that does not perform test 1 (re-exposure) after training and does not provide exercise tasks (hereinafter referred to as “first control group”)
(3) A group that did not perform test 1 (re-exposure) after training and was used for exercise tasks (hereinafter referred to as “second control group”)
(4) A group in which the time for returning to the chamber in test 1 (re-exposure) after training is 3 minutes and not used for exercise tasks (hereinafter referred to as “third control group”)
(5) The group returned to the chamber in test 1 (re-exposure) after training for 3 minutes, and then subjected to exercise tasks (hereinafter referred to as “fourth control group”)
(6) A group in which the time for returning to the chamber in test 1 (re-exposure) after training is 10 minutes and not used for exercise tasks (hereinafter referred to as “fifth control group”)

The results are shown in FIG. Each symbol shown in FIG. 18 represents the following values, and (A) to (F) represent the percentage of freezing time indicated by each group during training, and (G) to (J
) Is the percentage of freezing time indicated by each group during test 1 (re-exposure), and (K) through (P) are freezing time indicated by each group during testing (Freezing Time). ) Ratio.
(A) Proportion of freezing time indicated by the first control group (B) Proportion of freezing time indicated by the second control group (C) Freezing time indicated by the third control group Proportion of time (Freezing Time) (D) Proportion of freezing time indicated by the fourth control group (E) Proportion of freezing time indicated by the fifth control group (F) Running group The percentage of freezing time indicated by (G) The percentage of freezing time indicated by the third control group (H) The percentage of freezing time indicated by the fourth control group (I) Proportion of freezing time indicated by the fifth control group (J) Proportion of freezing time indicated by the running group (K) Shown by the first control administration group Proportion of freezing time (L) Proportion of freezing time indicated by second control administration group (M) Proportion of freezing time indicated by third control group (Freezing Time) N) Proportion of freezing time indicated by the fourth control group (O) Proportion of freezing time indicated by the fifth control group (P) Freezing time indicated by the running group (Freezing Time) Time)

As shown in FIG. 18, the percentage of freezing reaction time at test 1 (re-exposure) is not significantly different among all groups, and fear memory is formed after training, and fear memory is recalled by re-exposure. It has been confirmed. In addition, the ratio of freezing time shown by the fourth control group at the time of test 2 was the same value as that of the other control groups.
On the other hand, the ratio of freezing time shown by the running group at test 2 was significantly lower than that of the other groups.
In this Experimental Example 9, the first control group, the third control group and the fifth control group were 12 mice, the second control group was 9 mice, the fourth control group was 11 mice, and the running group was 11 mice. The results are shown as mean ± standard error. Comparison of each data was statistically analyzed using T test, and when p <0.05, it was judged that there was a significant difference.

  From the results of this experimental example 9, the old memory was brought back to the chamber for 3 minutes and recalled the fear memory, and even if it was subjected to an exercise task, it was not forgotten, but it was returned to the chamber for 10 minutes and recalled the fear memory for a long time. It was revealed that (i) the hippocampal dependence was revived, and (ii) it was later forgotten by exercise tasks.

  From the results of the experimental example 6 and the experimental example 9, the old memory is recalled for a long time of 10 minutes (i) “resurrection of hippocampal dependence”, and (ii) “neurogenesis in the hippocampus” It became clear that it was forgotten only by “prompt”.

  From the results of the above experimental examples, the screening method of the present invention converts the fear memory into a hippocampal-independent memory, for example, a non-human that has passed 8 weeks after being subjected to a fear conditioning context learning task (Ii) a test substance that promotes neurogenesis in the hippocampus after (i) reviving the hippocampal dependence of the fear memory, that is, gene expression in the hippocampus is induced by memory recall. And the change in behavioral characteristics can be analyzed to know that the test substance has efficacy as a therapeutic agent for post-traumatic stress disorder (PTSD).

  In addition, the substance tested by the screening method of the present invention is “promotes neurogenesis in the hippocampus” by administration after “restoring hippocampal dependence” of fear memory. As described above, continuous exposure therapy (PE) is an exposure therapy that reminds us of a shocking event that has become traumatic, and the process of recollecting fear memory in the fear conditioning contextual learning task (re-exposure). ) Is considered a model for the above-mentioned exposure (sustained exposure) therapy.

  Therefore, the substance tested according to the present invention can promote forgetting of fear memory when administered during continuous exposure therapy (PE), thereby leading to worsening post-traumatic stress disorder (PTSD). It is possible to suppress as much as possible. In addition, since the substance tested according to the present invention “promotes neurogenesis in the hippocampus”, it induces “re-immobilization” and “erasure” of memory like the conventional D-cycloserine. It does contribute to the treatment of post-traumatic stress disorder (PTSD) without the need to consider the timing at which it is present. For this reason, it is not necessary to determine the timing of the administration, and as a result, shortening of the treatment time of continuous exposure therapy (PE) can be achieved.

  The memantine is widely used as a therapeutic agent for Alzheimer's dementia. And from Example 5, it was revealed that memantine tested by the screening method of the present invention has an effect as a therapeutic agent for posttraumatic stress disorder (PTSD). From this, it can be said that the screening method according to the present invention is rich in practicality.

  The substance detected by the screening method according to the present invention can be a therapeutic agent for posttraumatic stress disorder (PTSD) in humans and animals.

Claims (5)

In a state where fear memory in a non-human animal subjected to a fear conditioning contextual learning task is converted into a hippocampal-independent memory, the non-human animal is reminded of the fear memory to induce gene expression in the hippocampus A first step of
A second step of administering a test substance that promotes neurogenesis in the hippocampus to the non-human animal after the first step, and
A screening method for a therapeutic agent for post-traumatic stress disorder, wherein the influence of a test substance on the non-human animal is detected using forgetting fear memory in the non-human animal as an index.   The screening method according to claim 1, wherein the first step is performed 8 weeks after the non-human animal is subjected to a fear conditioning context learning task.   The screening method according to claim 2, wherein in the first step, the non-human animal is returned to the chamber used for the fear conditioning context learning task for 10 minutes.   The screening method according to claim 3, wherein the gene is an early response gene.   The screening method according to claim 4, wherein the non-human animal is a rodent.

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