WO2013038666A1

WO2013038666A1 - Light-induced seizure model in rat brains

Info

Publication number: WO2013038666A1
Application number: PCT/JP2012/005805
Authority: WO
Inventors: 伸一郎大沢; 真樹岩崎; 元虫明; 寛八尾; 悌二冨永; 義人古澤; 浩史冨田; 隆一重本
Original assignee: 国立大学法人東北大学; 大学共同利用機関法人自然科学研究機構
Priority date: 2011-09-12
Filing date: 2012-09-12
Publication date: 2013-03-21
Also published as: JP6108469B2; JPWO2013038666A1

Abstract

The invention pertains to the use of a nonhuman mammal that expresses a photoreceptive protein in the nerve cells of the brain as a seizure model animal. More specifically, the invention pertains to a method for evaluating seizures using these model animals and a method for screening drugs to prevent or treat seizures, such as antiepileptic drugs.

Description

Rat brain light-induced convulsion model

The present invention relates to the use of a non-human mammal that expresses a photoreceptor protein in brain neurons as a convulsion model animal. More specifically, the present invention relates to a method for evaluating convulsions using the model animal and a method for screening for preventive or therapeutic agents for convulsions such as antiepileptic drugs.

Epilepsy is a relatively common neurological disease affecting approximately 1% of the population, but its biological origin has not been known for a long time. In epilepsy, cerebral neurons spontaneously fire in an abnormally synchronized manner, and the cerebral activity of that part is impaired, and symptoms appear, but the causes are diverse, and the mechanism of abnormality varies depending on the focal site. It was difficult to elucidate the fundamental mechanism.

So-called “epilepsy model animals” are used as a means for developing and developing epilepsy diagnosis and treatment methods. Many types of animal models have been reported that play an important role in elucidating the mechanism of acquired epileptogenicity, but it takes a long time of several weeks to several months to create it, like electrical stimulation in the brain. Things (Reference 1), those in which the status of convulsions persists, those that have not been able to reproduce epileptic seizures in humans that end intermittently within a few minutes (Reference 2), and those with a high mortality rate (Reference 2) ), There are cases where most of the similar creation techniques do not cause seizures and the model creation efficiency is poor (Reference 2), and there is a great limitation on the reproducibility of seizures and the arbitraryness of induction. Specifically, (i) administration of a neurotoxin (such as kainic acid) or metal (such as alumina gel) or physical stimulation (such as cerebral contusion) destroys specific parts and functions of the brain; (ii) intermittent Exciting by electrical stimulation (Kindling model, etc.), (iii) Gene modification, etc., all of which have the above drawbacks, and various studies have been conducted to date on the development of new models that have improved these drawbacks. Has been made.

On the other hand, in recent years, research using light-sensitive cation channels using genetic engineering has been active. Cell-selectivity, spatial-selectivity, and time-selectivity by expressing a photoreceptive protein in a target cell by gene transfer using a transgenic animal or viral vector, and stimulating at a specific wavelength from an optical fiber placed in the brain It has become possible to construct an experimental system that is significantly higher than before and that has smaller electrical artifacts. It has been reported that convulsive activity caused by deep brain stimulation (Reference 3, Reference 4) and electrical stimulation on brain slices can be suppressed by light stimulation (Model 5) for Parkinson's disease model animals. There is little research.

An object of the present invention is to provide a model animal that can induce a seizure attack immediately and with high reliability.

In order to achieve the above object, the inventors conducted research to establish a model animal experiment system that allows easy observation of seizure activity by creating a convulsion model using light stimulation, and collected it from the diatom Chlamydomonas. It was discovered that convulsive seizures can be induced in the rat by applying light stimulation to the hippocampus of a transgenic rat into which channel rhodopsin 2 (ChR2), which is a photoreceptive protein, has been introduced. Furthermore, the inventors have discovered that convulsive seizures can be induced in the rat by applying light stimulation to the hippocampus of the rat introduced with the ChR2 gene into the hippocampus by gene transfer using an adeno-associated virus-5 vector.

That is, the present invention is as follows.
(1) Use of a non-human mammal expressing a photoreceptor protein in brain neurons as a convulsion model animal.
(2) The use according to (1), wherein convulsions are induced by irradiating the hippocampus with light.
(3) The use according to (1) or (2), wherein the photoreceptor protein is a photosensitive cation channel.
(4) The use according to (3), wherein the photosensitive cation channel is channelrhodopsin 2.
(5) The use according to any one of (1) to (4), wherein the animal is a rodent.
(6) The use according to any one of (1) to (5), wherein the animal is an animal into which a gene for a photoreceptor protein has been introduced by infection with a viral vector.
(7) The use according to any one of (1) to (5), wherein the animal is a transgenic animal into which a gene for a photoreceptor protein has been introduced.
(8) The use according to (7), wherein the animal is a transgenic rat in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a mutant thereof that exhibits convulsions upon irradiation with light.
(9) A method for evaluating seizures, comprising a step of using a non-human mammal expressing a photoreceptive protein in brain neurons and irradiating the hippocampus of the animal with light.
(10) The method according to (9), further comprising a step of monitoring an electroencephalogram.
(11) The method according to (9) or (10), wherein the photoreceptor protein is a photosensitive cation channel.
(12) The method according to (11), wherein the photosensitive cation channel is channelrhodopsin 2.
(13) The method according to (12), wherein the wavelength of light is 420 to 520 nm.
(14) The method according to (12) or (13), wherein the light intensity is 10 to 120 mW.
(15) The method according to any one of (12) to (14), wherein the frequency of light is 5 to 40 Hz, or the light is continuously irradiated.
(16) The method according to any one of (12) to (15), wherein the light pulse width is 0.5 ms to continuous.
(17) The method according to any one of (12) to (16), wherein the light is intermittently irradiated so that the on / off ratio is 0.02 to 0.9.
(18) The method according to any one of (9) to (17), wherein the animal is a rodent.
(19) The method according to any one of (9) to (18), wherein the animal is an animal into which a gene for a photoreceptor protein has been introduced by infection with a viral vector.
(20) The method according to any one of (9) to (18), wherein the animal is a transgenic animal into which a gene for a photoreceptor protein has been introduced.
(21) The method according to (20), wherein the animal is a transgenic rat in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a mutant thereof that exhibits convulsions upon irradiation with light.
(22) A convulsion model non-human mammal in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a variant that exhibits convulsions upon irradiation with light.
(23) A method for screening a prophylactic or therapeutic agent for seizures including epilepsy using a non-human mammal that expresses a photoreceptor protein in brain neurons.
(24) A test substance is administered to an animal, and the induction rate of convulsion induced by light irradiation and / or the duration of convulsions are compared and evaluated as compared with the case where no test substance is administered. 23) The screening method according to 23).

Transgenic non-human mammals that express photoreceptor proteins in brain neurons used in the present invention and non-human mammals in which photoreceptor protein genes are introduced into brain neurons by infection with viral vectors respond to light irradiation And convulsive seizures are triggered immediately and reliably. Therefore, the animal can be used as a model animal for seizures including epilepsy. In addition, the model animal can be used to screen for preventive or therapeutic agents for convulsions such as antiepileptic drugs.

FIG. 1A is a diagram schematically showing a DNA fragment to be introduced into a rat. FIG. 1B shows the expression pattern of the introduced gene. FIG. 2 is a diagram schematically showing the method of the present invention. FIG. 3 is a chart showing neural activity during electrical stimulation and light stimulation. FIG. 4 is a diagram schematically showing the intermittent light stimulation method. FIG. 5 is a chart showing electroencephalograms measured in a convulsion model rat during and after light stimulation. FIG. 6 shows the results of examining the influence of the on / off ratio and frequency on the seizure induction rate. FIG. 7 is a diagram showing the seizure induction rates of the hippocampus, amygdala, prothalamic nucleus, and sensorimotor cortex when light stimulation is applied. FIG. 8A is a graph showing changes in seizure induction rate and seizure duration when diazepam is administered. FIG. 8B shows changes in convulsive activity when diazepam is administered. FIG. 9A is a schematic diagram showing an experimental protocol in which light stimulation is given to the rat hippocampus into which a channelrhodopsin gene has been introduced using a viral vector. FIG. 9B is a photomicrograph confirming the expression of the introduced ChR2V. FIG. 9C is a chart showing electroencephalograms measured in a convulsion model rat during and after light stimulation.

1. Definitions A “photoreceptive protein” is a protein that exhibits some response by absorbing light of a specific wavelength, and includes sensory rhodopsin involved in light-sensitive ion channels and signal transduction. Here, the “photosensitive ion channel” is a photosensitive channel protein in which a channel opens and closes by light stimulation of a specific wavelength, and depolarization or hyperpolarization occurs due to ion migration. Photosensitive cation or anion channels combine the functions of a photoreceptor and the function of a cation channel or anion channel in a single molecule, and convert light brightness information into electrical information. Can do.

Photoreceptive proteins that can be used in the present invention include channelrhodopsins, which are photosensitive cation channels. The photosensitive cation channel is not particularly limited as long as it is a photosensitive cation channel found in Chlamydomonas or other organisms and a gene variant thereof. For example, channel rhodopsin 2 (ChR2) (SEQ ID NO: 1), channelrhodopsin 1, and modified channelrhodopsin. In particular, it is possible to use a variant with enhanced efficiency in causing an action potential caused by a light pulse by adding a property having a property of accumulating to a specific location of a neuron in the intracellular domain. In addition, variants with different ion permeability, for example, variants with enhanced Ca ²⁺ permeability, K ⁺ selective or anion selective permeability, and negative displacement of membrane potential by light stimulation ( It is also conceivable to use a variant that causes hyperpolarization. In addition, it is possible to use variants having different absorption spectra.

The ChR2 protein encoded by the ChR2 gene is not limited to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, but substitution or deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1, Those having an addition or insertion and having a biological activity equivalent to or higher than that of the ChR2 protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or having substantially the same sequence homology as the amino acid sequence shown in SEQ ID NO: 1 And having a biological activity equivalent to or higher than that of the ChR2 protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as “ChR2 mutant polypeptide”).

In the present specification, “several” used in the context of ChR2 mutant polypeptide is an integer of 10 or less, for example, an integer of 2 to 9, 2 to 7, or 2 to 5.

The “substantially identical sequence homology” is at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% of the amino acid sequence shown in SEQ ID NO: 1. It means having homology. The% homology refers to a value calculated using software (for example, FASTA, DNASIS, BLAST, etc.) that calculates the homology between a plurality (two) of amino acid sequences with default settings.

In the present specification, “equivalent biological activity” means that biological activities such as photosensitivity and channel function have substantially the same strength. The term may also include the case of having substantially the same biological activity, and the “same” biological activity here is the same in light sensitive wavelength, for example, the nature of the ion permeable activity. Say something.

Further, the ChR2 gene is not limited to the polynucleotide comprising the base sequence shown in SEQ ID NO: 2, but is a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide comprising the base sequence shown in SEQ ID NO: 2. And a polynucleotide encoding a polypeptide having a biological activity equal to or higher than that of the ChR2 protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (hereinafter also referred to as “ChR2 mutant polynucleotide”). A person skilled in the art can appropriately combine the above or other factors that determine the stringency of hybridization (for example, the concentration, length and GC content of the hybridization probe, the reaction time of hybridization, etc.) Similar stringency can be achieved.

“Epileptic” refers to the same type of clinical seizures (systemic tonic-clonic seizures, absence seizures, hallucinogenic seizures, one-limbed seizures) within the same individual due to excessive synchronous firing of cerebral neurons. This refers to a pathological condition that repeats tonic seizures in the club. Epilepsy is a neurological disease of unknown biological origin and is associated with convulsions.

Here, “convulsions” refers to involuntary and seizure contractions of the muscle groups of the whole body or part of the body accompanied by characteristic brain wave changes. In the present specification, a state in which convulsions are spontaneously and repeatedly induced is defined as epilepsy, and the terms seizure, seizures, seizure activity, and seizures are all presenting with seizures. It shall be said.

In this specification, “intermittently irradiating light” means, as shown in FIG. 4, that light is continuously emitted for a certain time (pulse width or on-time) with a certain time interval (stimulation interval or off-period). (Period) irradiation time (stimulation period), and the period of non-irradiation after the stimulation period (non-stimulation period) means repeating, such light irradiation method is defined as intermittent light stimulation method . In the stimulation period, light irradiation with a pulse width (on period) is repeated with a stimulation interval (off period), and the repetition is performed at a frequency (Hz) (1) with the pulse width irradiation and the stimulation interval as one cycle. The number of cycles per second). Furthermore, the ratio of the pulse width in one cycle (pulse width / pulse width + stimulation interval) is defined as “on / off ratio”.

2. Seizure model non-human mammal (1) Production of model animal (a) Production of transgenic animal In the present invention, a transgenic non-human mammal that expresses a photoreceptor protein in brain neurons is used. Such an animal is prepared by introducing a gene of a photoreceptor protein into a non-human mammal and expressing the gene specifically in brain neurons according to a method for producing a transgenic animal well known to those skilled in the art. Can do.

Specifically, although not limited, for example, a transgenic animal can be produced by the following steps. A gene of a photoreceptor protein is introduced into an expression vector for a non-human mammal cell having an expression control sequence that functions in the brain neurons of the non-human mammal. A linear DNA fragment containing the transgene, linearized from this gene expression vector, is introduced into a totipotent cell derived from a non-human mammal such as a rat, and transplanted to a temporary parent after the introduction. Used in the present invention by generating individuals from transplanted totipotent cells and selecting individuals in which the transgene is integrated into the somatic chromosome by PCR or the like using DNA extracted from the tissue of the individual Transgenic animals can be obtained. A preferred example of the introduction of a DNA fragment into a totipotent cell is a microinjection method. Examples of totipotent cells used for producing a transgenic animal include cultured cells such as fertilized eggs and early embryos, ES cells having multipotency, and the like.

The “expression control sequence that functions in the brain neurons of the non-human mammal” is not particularly limited as long as it is an expression control sequence that functions in the brain neurons of the non-human mammal, but in the brain neurons of the non-human mammal. It is preferably an expression control sequence that functions specifically, and more preferably a promoter that functions specifically in brain neurons of a non-human mammal. As a nerve cell-specific promoter that functions in a non-human mammal, for example, Thy1, 2 region, CaMKII, PV and the like can be used.

By crossing individuals whose transgenes have been confirmed, transgenic animals in which the photoreceptive protein used in the present invention has been incorporated into a part of the chromosome so that it can be stably expressed in brain neurons can be efficiently produced. can do.

The non-human mammal used in the present invention may be a mammal other than a human, and examples thereof include rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, monkeys and the like. Preferably, rodents such as rats, mice, hamsters and guinea pigs are used, and more preferably rats and mice are used.

In order to confirm the expression of the introduced photoreceptor protein gene, a gene encoding a fluorescent protein may be introduced together. In the present invention, fluorescent proteins well known to those skilled in the art can be used. For example, GFP, YFP, RFP, DSRed, and Venus may be used. This makes it possible to identify nerve cells that respond to light stimulation by confirming fluorescence in the tissue.

In order to produce a transgenic animal for use in the present invention, a gene sequence encoding a photoreceptive protein, and optionally a fluorescent protein, is encoded under the control of an expression control sequence that functions in brain neurons of a non-human mammal. An expression vector for non-human mammalian cells in which the sequence of the gene to be ligated in such a manner that it can function is used. Examples of expression vectors for non-human mammalian cells include pEGFP-C1 (Clontech), pGBT-9 (Clontech), pcDNAI (Invitrogen), pcDNA3.1 (Invitrogen), pEF- BOS (Nucleic® Acids® Res., 18, 5322, 1990), pAGE107 (Cytotechnology, 3, 133, 1990), pCDM8 (Nature, 329, 840, 1987), pcDNAI / AmP (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen) ), PAGE103 (J. Blochem., 101, 1307, 1987), pAGE210, and the like.

As a suitable example of the above-mentioned transgenic animal, a transgenic rat identified by the receipt number NITE ABP-1417 described later can be mentioned.

(B) Gene transfer by viral vector In the present invention, a non-human mammal that expresses a photoreceptor protein in brain neurons by gene transfer using a viral vector can also be used. Gene transfer into animals using viral vectors can be performed according to methods well known to those skilled in the art.

Specifically, although not limited, for example, the gene can be introduced in vivo by the following steps. First, under the control of an expression control sequence, a viral vector having a sequence in which a gene sequence encoding a photoreceptor protein and optionally a sequence of a gene encoding a fluorescent protein are linked in a functional manner is prepared.

In the present invention, the “viral vector” is not particularly limited, and refers to a vector for gene transfer utilizing a mechanism for infecting or maintaining a virus cell, and includes any known viral vector. In the present invention, for example, a Sendai virus vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, and the like can be used. In the present invention, the “expression control sequence” is not particularly limited, and an enhancer and / or promoter sequence well known to those skilled in the art can be used. For example, calmodulin kinase II, parvalbumin promoter, etc. can be used.

A gene encoding a fluorescent protein may be arbitrarily introduced in order to confirm the expression of the introduced photoreceptor protein gene. In the present invention, fluorescent proteins well known to those skilled in the art can be used. For example, GFP, YFP, RFP, DSRed, and Venus may be used. This makes it possible to identify nerve cells that respond to light stimulation by confirming fluorescence in the tissue.

A virus vector containing a photoreceptive protein gene sequence is injected into a target cell, target tissue or target organ (hereinafter referred to as “target cell etc.”). In the present invention, “target cell”, “target tissue”, and “target organ” refer to any cell existing in the brain, any site in the brain, and brain, respectively. In the present invention, the viral vector is preferably injected into the hippocampus. A virus is infected by injecting a viral vector into a target cell or the like, and a gene encoding a photoreceptor protein gene and a fluorescent protein is introduced into the cell.

(2) Seizure induction (light irradiation process)
In the present invention, in order to induce convulsions in the transgenic animals and animals transgenic using the viral vectors, the animals are opened, and the brain, particularly the hippocampus, is intermittently irradiated with light to stimulate light. Do. The on / off ratio of light irradiation is 0.02 to 0.9 or continuous stimulation, preferably 0.05 to 0.5, more preferably 0.05 to 0.1. The pulse width may be 0.5 ms to continuous, preferably 0.1 to 100 ms, more preferably 0.1 to 50 ms. In the present invention, it is possible to induce convulsions, albeit at a low frequency, by irradiating the transgenic animal with light continuously rather than intermittently.

The wavelength of the light irradiated in the method according to the present invention is about the response wavelength of photoreceptive protein ± 50 nm, preferably ± 35 nm, more preferably about ± 20 nm. Therefore, when channelrhodopsin 2 that is depolarized by light having an optimum wavelength of 470 nm is used as the photoreceptive protein, light having a wavelength of about 420 to 520 nm, preferably about 435 nm to 505 nm, more preferably about 450 to 490 nm. Irradiate.

The intensity of the irradiated light is about 10 to 120 mW, preferably about 30 to 100 mW, more preferably about 50 to 80 mW.

The frequency of the irradiated light is about 5 to 40 Hz, preferably about 10 to 20 Hz.

Transgenic animals and animals that have been transfected with viral vectors exhibit convulsions when the hippocampus is subjected to light stimulation, in particular light stimulation according to the intermittent light stimulation method. It is considered that depolarization is caused in a nerve cell expressing a light-sensitive cation channel by light stimulation, so that a transgenic animal and an animal transgenic with a viral vector exhibit convulsions.

In the present invention, in order to induce convulsions in a transgenic non-human mammal incorporating a photoreceptive protein gene and a non-human mammal introduced with a viral vector, (a) a light source, (b) a light source An apparatus is used that includes coupled photoconductive leads and (c) means for measuring brain waves. The apparatus may further include (d) means for data processing of the measured electroencephalogram and / or (e) means for displaying the measured electroencephalogram.

The light source that can be used in the apparatus is not particularly limited as long as it emits light having a response wavelength of the photoreceptive protein. For example, a laser light source is used.

A photoconductive conductor that can be used in the device is a means for conducting light or light energy from one location to another, such as an optical fiber.

As a means for measuring an electroencephalogram that can be used in the apparatus, an electroencephalograph known in the art can be used.

The above apparatus is used together with the model animal according to the present invention for evaluation of convulsions, screening for preventive or therapeutic drugs for convulsions described later.

(3) Evaluation of convulsions (animal observation process or EEG monitoring process)
Seizures can be detected by observing transgenic animals and animals transfected with viral vectors and measuring brain waves. For example, when vibration occurs in the beard, limbs, trunk, or tail, or when abnormal brain waves are observed, it is determined that convulsion has occurred. In this specification, an abnormal electroencephalogram is an activity characterized by spontaneous continuous sharp waves and spikes, which are induced by intermittent light stimulation, but are generated independently of each evoked potential. Activity.

Transgenic animals that have been subjected to light stimulation and animals that have been transfected with a viral vector exhibit a latent period that does not exhibit convulsions even when irradiated with light, and then a convulsions (Sz) period that exhibits convulsions (FIG. 4). And FIG. 5). The convulsion period continues for a certain period of time even after the light stimulation is finished, but eventually ends spontaneously. In this specification, the incubation period and the convulsion period are combined and defined as one session.

Thus, in the present invention, it is possible to more easily and accurately determine the presence or absence of convulsive seizure induction in a model animal by measuring an electroencephalogram.

Electroencephalogram monitoring can be performed according to a method known in the art. Specifically, a deep electrode is inserted into the brain of a model animal, and the electrode is connected to an electroencephalograph to measure an electroencephalogram. Therefore, in the method of the present invention, the deep electrode is preferably inserted into the hippocampus. While the seizures are induced, EEG findings are measured only for the evoked potential for each stimulus in the early stage of the stimulus, but the waveform gradually changes and spontaneous seizure activity occurs from a certain point. At the end of the stimulus, it continuously shifts to autonomous seizure activity. This activity can be observed throughout the induction period in this model with few artifacts (see FIGS. 3 and 5).

3. Seizure model rat In the present invention, a transgenic non-human mammal that expresses a photoreceptor protein in brain neurons is used. In one embodiment of the present invention, a transgenic rat expressing Chlamydomonas-derived channelrhodopsin 2 specifically in brain neurons can be used.

(1) Deposit of fertilized egg The fertilized egg of a transgenic rat introduced with channelrhodopsin 2, which can be used in the present invention, is a microorganism identification display Thy1.2-ChR2-Venus, September 2012 On the 4th (reception date), it was deposited with the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818). The receipt number is NITE ABP-1417.

(2) Characteristics Thy1.2-ChR2-Venus has been introduced into the deposited transgenic rat. The transgenic rat exhibits convulsions when the hippocampus is irradiated with light according to the intermittent light stimulation method. However, in the transgenic rats, the cortex and amygdala, which have been considered to be prone to convulsions by conventional stimulation, and the prethalamic nucleus, which is thought to play an important role in the network of seizure activity of epilepsy, are similarly illuminated. Adding a stimulus does not induce a seizure (Figure 7).

(3) Breeding / breeding conditions The deposited transgenic rats can be proliferated by brother-sister mating to ensure the necessary number of rats, as is usually used for breeding inbred animals. The breeding conditions are not particularly limited, and can be raised according to a conventional breeding method.

(4) Offspring, congenic lines, and mutants Transgenic rats that can be used in the present invention have not only the transgenic rat that has been deposited, but also the introduced gene, that is, the ChR2 gene (SEQ ID NO: 2). Also included are crossed rats obtained by crossing the transgenic rats and experimental rats that exhibit the above characteristics, and congenic rats. Also included are mutants of the transgenic rat that express the ChR2 mutant polypeptide specifically in brain neurons and exhibit the characteristics described above. In addition, the congenic rat has a genetic background other than the introduced gene of that of any experimental rat, and such a congenic rat is a transgenic rat that has been deposited. In addition, a transgenic rat that is heterozygous or homozygous for the ChR2 gene and any experimental rat can be produced by proceeding backcrossing according to a known backcross breeding method.

(5) Usefulness and Advantages The convulsion model animal of the present invention is superior to the conventional epilepsy model animal in the following points.
・ Because it shifts to convulsive activity as it is after light stimulation, it does not take time to induce convulsive seizure. ・ Because convulsive seizure can be induced immediately after placing optical fiber and electrode, it takes a long time to create a model such as kindling model. No convulsive seizures can be triggered arbitrarily at multiple times within minutes to hours, and reproducibility is very high within or between individuals.Light stimulation has few electrical measurement artifacts. Therefore, it is convenient to measure and analyze epilepsy activity, which is an abnormality of electrical activity. ・ The convulsion model animal of the present invention has a lower mortality rate than the conventional model animal. ・ Receive number NITE used in the present invention. ABP-1417 transgenic rats, and their progeny, congenic rats, and mutants Identification (genotyping) is easy. Typing uses a somatic cell (tail) sample to detect the Venus (not present in the Wild Wister rat gene), which is part of the recombinant gene, by the PCR method. Receipt number used in the present invention NITE ABP-1417 transgenic rats, and their progeny, congenic rats, and mutants exhibit 100% seizures, regardless of whether they are heterozygous or homozygous.

4). Drug screening method and deep brain stimulation therapy using convulsion model non-human mammal (1) Drug screening method The convulsion model non-human mammal of the present invention can be used as an epilepsy model non-human mammal. Therefore, a drug for preventing or treating convulsions including epilepsy can be screened by administering a test substance to the convulsion model non-human mammal of the present invention.

For example, a test substance is administered to the model animal of the present invention, an index value correlated with convulsions in the model animal or a part thereof is measured, and compared with the result of a control animal not administered the test substance. Based on the comparison result, by confirming whether or not to reduce or eliminate convulsions, a prophylactic or therapeutic agent for convulsions including epilepsy can be screened.

The model animal or a part thereof includes both the whole body of the animal and a limited tissue or organ. In the case of limited tissues or organs, it includes those extracted from animals.

As the index value, for example, changes in the duration of convulsions and the induction rate can be used. In the present specification, “duration of convulsions” refers to the time during which a convulsion model animal that has received light stimulation exhibits convulsions, and is also referred to as a convulsion period. Further, in the present specification, “convulsion induction rate” refers to the frequency of occurrence of convulsions in a convulsion model animal that has been subjected to light stimulation, and the model animal exhibited convulsions relative to the number of times of light stimulation. The number of times is expressed as a percentage.

For example, when a decrease in the duration and / or induction rate of convulsions is observed by administration of a test substance, the test substance used can be selected as a prophylactic or therapeutic agent for seizures including epilepsy.

Also, changes in vibrations in the whiskers, limbs, trunk, or tail, or changes in abnormal brain waves can be used as index values. For example, when disappearance or alleviation of convulsions or normalization of abnormal electroencephalograms is observed by administration of a test substance, the test substance used can be selected as a prophylactic or therapeutic agent for seizures including epilepsy.

Examples of test substances include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammalian tissue extracts, plasma, etc. The compound may be a novel compound or a known compound. These test substances may form a salt, and as a salt of the test substance, a salt with a physiologically acceptable acid (for example, inorganic acid) or a base (for example, organic acid) is used. . Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

The administration method of the test substance can be appropriately selected by those skilled in the art depending on the nature of the substance, and for example, oral administration, intravenous injection, transdermal administration, subcutaneous administration, intradermal administration, intraperitoneal administration, etc. are used. . The dosage of the test substance can be appropriately set by those skilled in the art depending on the administration method and the nature of the substance.

(2) Deep brain stimulation therapy As described above, the convulsive model non-human mammal of the present invention can also be used as an epilepsy model non-human mammal. Therefore, convulsions including epilepsy are prevented or treated by applying physical stimulation such as electricity to the brain of the convulsion model non-human mammal of the present invention, imitating deep brain stimulation (DBS). Therefore, it is possible to identify a region in the brain where a physical stimulus effectively acts, a stimulation parameter, and the like. Here, deep brain stimulation therapy is a method of causing some change in activity in the brain by applying electrical stimulation from electrodes placed deep in the brain, for treating symptoms such as epilepsy and Parkinson's disease It can also be a surgical procedure.

For example, imitating deep brain stimulation therapy, placing a stimulation electrode in the brain of the model animal of the present invention, applying physical stimulation such as electrical stimulation, etc., and correlating with convulsions in the model animal or a part thereof An index value having Including epilepsy by comparing with the results of control animals not receiving physical stimulation or model animals prior to physical stimulation and confirming whether or not convulsions are reduced or eliminated based on the comparison results Sites and stimulation parameters in the brain where physical stimulation can effectively work in the prevention or treatment of seizures can be identified.

For example, electrical stimulation and electrical and chemical cauterization can be used as physical stimulation. In addition, as the index value, as in the above (1) drug screening method, for example, changes in convulsion duration and induction rate, changes in vibration in the beard, limbs, trunk, or tail, or changes in abnormal electroencephalogram Can be used. For example, if a decrease in the duration and / or induction rate of convulsions is observed by electrical stimulation, or if disappearance or reduction of convulsions, or normalization of abnormal electroencephalograms is observed, prevention or treatment of seizures including epilepsy It is possible to identify a site in the brain where a physical stimulus effectively works and a stimulation parameter.

The contents of all patents and references explicitly cited herein are hereby incorporated by reference. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2011-198126 (filed on September 12, 2011), which is the application on which the priority of the present application is based, are incorporated herein by reference. It is.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention.

1. System construction (1) Transgenic rat Thy1.2-ChR2-Venus transgenic rat prepared by incorporating Channelrhodopsin-2 (ChR2) and Venus under the mouse Thy1.2 promoter using gene recombination technology to Wister rat. Use. The Thy1.2 promoter is a promoter region that is selectively expressed in thymic stromal cells and neurons, ChR2 is the above-described photoreceptor cation channel, and Venus is a fluorescent protein. In this rat neuron, ChR2-Venus fusion protein is expressed, and the expression distribution of ChR2 can be evaluated by observing the fluorescence of Venus.

The transgenic rat used in this experiment was created by the research of Prof. Hiroshi Yao, Graduate School of Medicine, Tohoku University and Professor Ryuichi Shigemoto, National Institutes of Natural Sciences, receiving number NITE ABP-1417 .

(2) Experimental design (a) Apparatus The apparatus used consists of a rat fixation device, an electroencephalogram measurement device, and a light stimulation device.
(B) Craniotomy Transgenic rats are opened under anesthesia, and electrodes are placed in any part of the brain where the electroencephalogram is to be measured, and an optical fiber is placed in the part and direction where light reaches the hippocampus (see FIG. 2). ).
(C) Neural activity visualization An electroencephalogram is recorded by an electrode placed in the brain (see FIG. 3).
(D) Intermittent light stimulation method The light stimulation uses the above-mentioned intermittent stimulation. The stimulation frequency and the on / off ratio can be arbitrarily changed (see FIG. 4).

(3) Light-induced seizure activity When photostimulation is performed under electroencephalogram measurement, photocurrent due to photostimulation is measured on the electroencephalogram, but spontaneous seizure activity gradually occurs and grows. In addition, clastic convulsions are recognized as physical findings such as beards and extremities. This activity continues to spontaneous seizure activity after the end of light stimulation, and eventually ends. Recording of the electroencephalogram is possible throughout (see FIG. 5).

(4) Optimum conditions for inducing convulsions In the hippocampus, the frequency of convulsions was measured under conditions of 5-40 Hz, on / off ratio 0.02-0.9, and continuous stimulation, and 10-20 Hz, on / off. The induction frequency was maximum (10/10) times under the stimulation condition with an off ratio of 0.05-0.1 (see FIG. 6). In addition, when stimulating convulsions by applying light stimulation to the hippocampus, amygdala, anterior thalamus, and sensorimotor cortex under a stimulation condition of 10 Hz and an on / off ratio of 0.1, the induction is mostly made outside the hippocampus. None (see FIG. 7).

2. Changes in convulsions induced by diazepam administration (1) Experimental design Transgenic rats were divided into two groups, diazepam administration group and phosphate buffered saline administration group (control), and diazepam administration group had diazepam 10 mg / ml / kg body weight. (0.2-0.4 ml) was administered intraperitoneally to the control group at 1 ml / kg body weight of phosphate buffered saline. Before and after administration, convulsions were induced by light stimulation to the hippocampus 10 times every 5 minutes, and the induction frequency (induction rate) and convulsion duration were compared. The light stimulation conditions were a light intensity of 50 mW, a frequency of 10 Hz, a pulse width of 10 ms, and a total stimulation time of 10 seconds.

(2) Results The induction frequencies of the diazepam administration group and phosphate buffered saline administration group were (before administration, after administration), respectively, the diazepam administration group (70%, 20%), phosphate buffered saline administration group (90%, 90%) (see FIG. 8A). The average duration [seconds] (before and after administration) when convulsions occurred was the diazepam administration group (42.3, 6.6) and the phosphate buffered saline administration group (54.0, 61.1). (See FIG. 8A).

(3) Effect of diazepam administration In the diazepam administration group, the mean power [mV ² / sec] of convulsive activity calculated from the electroencephalogram was compared before and after administration, after administration (0.55 ± 0.7), after administration ( 0.11 ± 0.21), and there was a significant difference between the two (see FIG. 8B). Here, the power score in FIG. 8B is obtained by squaring the amplitude of the electroencephalogram and is an index representing the intensity of convulsive activity. From these results, it was considered that the seizure activity was suppressed by the administration of diazepam.

3. Induction of convulsions after photostimulation in hippocampus transfected with rAAV-ChR2-Venus vector (1) Preparation of AAV vector with ChR2 gene construct N-terminal fragment of ChR2 (GenBank accession number AF461397) (residues 1-315) Was fused in-frame to the fluorescent protein Venus at the end of the ChR2-coding fragment (ChR2V). The ChR2 gene was introduced into the EcoRI and HindIII sites of the 6P1 plasmid. The synapsin promoter was replaced with a hybrid cytomegalovirus enhancer / chicken β-actin promoter to construct AAV-ChR2V. AAV-RC and p-helper plasmids were obtained from Stratagene (La Jolla, CA, USA). The high titer (1-10 × 1012 particles / ml) rAAV vector (rAAV-ChR2V) was obtained by a previously reported method (Gene Therapy, 2011;
18 (3): 266-74).

(2) Induction of seizure Repeated light stimulation was given to the hippocampus of wild-type Wister rats transfected with rAAV-ChR2-Venus vector (FIG. 9A). AAV with a gene encoding a ChR2V fusion protein driven by a hybrid cytomegalovirus enhancer / chicken β-actin promoter was injected into the dentate hilus of normal rat septal hippocampus . After 4-6 weeks, strong expression of ChR2V was confirmed in hippocampal neurons (FIG. 9B). Repetitive pulsed light stimulation combined with parameters of frequency (5, 10, 20 Hz) and duty ratio (0.05, 0.1, 0.2) under simultaneous recording of LFP using hybrid electrodes Gave. Repeated light stimulation successfully induced convulsions in a reproducible manner (FIG. 9C).

The model animal prepared according to the method of the present invention is useful for elucidating the mechanism of convulsions, screening for anticonvulsants and antiepileptic drugs, and determining the therapeutic effect.

Claims

Used as a convulsion model animal for non-human mammals that express photoreceptor proteins in brain neurons.
Use according to claim 1, characterized in that convulsions are induced by irradiating the hippocampus with light.
Use according to claim 1 or 2, wherein the photoreceptor protein is a photosensitive cation channel.
Use according to claim 3, wherein the photosensitive cation channel is channelrhodopsin 2.
The use according to any one of claims 1 to 4, wherein the animal is a rodent.
The use according to any one of claims 1 to 5, wherein the animal is an animal into which a gene for a photoreceptor protein has been introduced by infection with a viral vector.
The use according to any one of claims 1 to 5, wherein the animal is a transgenic animal into which a gene for a photoreceptor protein has been introduced.
The use according to claim 7, wherein the transgenic animal is a transgenic rat in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a mutant thereof that exhibits convulsions upon irradiation with light.
A method for evaluating seizures, which comprises a step of using a non-human mammal expressing a photoreceptive protein in brain neurons and irradiating the hippocampus of the animal with light.
The method according to claim 9, further comprising a step of monitoring an electroencephalogram.
The method according to claim 9 or 10, wherein the photoreceptor protein is a photosensitive cation channel.
The method according to claim 11, wherein the photosensitive cation channel is channelrhodopsin 2.
The method according to claim 12, wherein the wavelength of light is 420 to 520 nm.
The method according to claim 12 or 13, wherein the light intensity is 10 to 120 mW.
The method according to any one of claims 12 to 14, wherein the frequency of light is 5 to 40 Hz, or light is continuously irradiated.
The method according to any one of claims 12 to 15, wherein the pulse width of the light is 0.5 ms to continuous.
The method according to any one of claims 12 to 16, wherein the light is intermittently irradiated so that the on / off ratio is 0.02 to 0.9.
The method according to any one of claims 9 to 17, wherein the animal is a rodent.
The method according to any one of claims 9 to 18, wherein the animal is an animal into which a gene for a photoreceptor protein has been introduced by infection with a viral vector.
The method according to any one of claims 9 to 18, wherein the animal is a transgenic animal into which a gene for a photoreceptor protein has been introduced.
21. The method according to claim 20, wherein the animal is a transgenic rat in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a mutant thereof that exhibits convulsions upon irradiation with light.
A convulsion model non-human mammal in which a fertilized egg is deposited under the receipt number NITE ABP-1417, or a variant that exhibits convulsions upon irradiation with light.
A method of screening for a prophylactic or therapeutic agent for seizures including epilepsy using a non-human mammal that expresses a photoreceptor protein in brain neurons.
24. The test substance is administered to an animal, and the induction rate and / or the duration of convulsions induced by light irradiation are compared and evaluated as compared with the case where no test substance is administered. The screening method described.