Method for evaluating the neurological effects of test substances, kit therefor, and neuronal cell culture vessel

Abstract

The present invention addresses the problem of evaluating a neural action of a test substance at a high sensitivity, high quantitativeness particularly in a low concentration region and high reproducibility.　This problem can be solved by: a method that successively comprises (A) a step for culturing cultured nerve cells on the surface of a molded body which is formed of a norbornene-based polymer and at least the surface of which is hydrophilized, (B) a step for bringing the cultured nerve cells into contact with a test substance, (C) a step for fixing the cultured nerve cells, (D) a step for visualizing drebrin clusters of dendritic spines of the cultured nerve cells, and (E) a step for measuring the line density along the dendrites of the drebrin clusters, and determining that the test substance has a neural action when the line density increases or decreases compared with the line density of cultured nerve cells which are not contacted with the test substance; and a kit and a culture container to be used in this method.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the neural activity of a test substance using cultured nerve cells, a kit for evaluating the neural activity of a test substance, and a nerve cell culture vessel for evaluating the neural activity of a test substance. [Background technology]

[0002] In order to stably culture adherent cells such as epithelial cells, they usually need to be attached to a culture vessel. For this reason, in order to improve the adhesiveness of cells to the vessel, the surface of a plastic culture vessel is usually hydrophilized by plasma treatment, gamma ray treatment, UV excimer treatment, etc. By performing the hydrophilization treatment, proteins such as extracellular matrix proteins, such as collagen, necessary for cell adhesion, can be easily coated on the surface of the culture vessel, enabling stable cell culture. The present inventors have disclosed that a molded article made of a norbornene-based polymer can be used as a material for a culture vessel suitable for culturing adherent cells (Patent Documents 1 and 2).

[0003] In patients with Alzheimer's disease, abnormal proteins such as amyloid beta accumulate in the brain, activating N-Methyl-D-aspartate (NMDA) glutamate receptors in the brain, and excessive activation of these receptors is thought to result in damage to nerve cells and impaired memory and learning functions.

[0004] The present inventors have disclosed that the NMDA-type glutamate receptor inhibitory activity of phencyclidine on hippocampal neurons derived from healthy rats can be confirmed by observing changes in drebrin-positive synapses and drebrin accumulation that occur when phencyclidine is applied to cultured neurons followed by the addition of glutamate (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Patent Publication No. 2015 / 199117 [Patent Document 2] International Patent Publication No. 2017 / 104618 [Non-patent literature]

[0006] [Non-Patent Document 1] J. Pharmacological and Toxicological Methods,99 (2019) 106583 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a highly sensitive and simple method for evaluating the neural activity of a test substance, a kit for use in evaluating the neural activity of a test substance, and a nerve cell culture vessel for use in evaluating the neural activity of a test substance. [Means for solving the problem]

[0008] Based on the disclosure of Non-Patent Document 1, the present inventors believed that changes in drebrin-positive synapses and drebrin accumulation could be used to evaluate the neural effects of test substances. Here, hippocampal neurons derived from Alzheimer's disease model mice have excessively activated NMDA-type glutamate receptors, resulting in reduced glutamate responsiveness compared to hippocampal neurons derived from healthy mice. Therefore, the present inventors believed that if hippocampal neurons derived from Alzheimer's disease model mice were cultured for a long period of time using commercially available hydrophilized polystyrene culture vessels and exposed to glutamate, changes in drebrin-positive synapses and drebrin accumulation compared to hippocampal neurons derived from healthy mice could be confirmed. However, in reality, it was found that no differences in drebrin-positive synapses and drebrin accumulation (drebrin clusters) could be confirmed between hippocampal neurons derived from Alzheimer's disease model mice and hippocampal neurons derived from healthy mice. As a result of extensive research, the present inventors have found that when a hydrophilized culture vessel made of cycloolefin polymer (COP), a type of norbornene polymer, is used, it exhibits adhesive properties suitable for neuronal culture, while also enabling detection of changes in drebrin-positive synapses and drebrin accumulation when glutamate is applied to hippocampal neurons derived from Alzheimer's disease model mice, compared to hippocampal neurons derived from healthy mice.Furthermore, they have found that such a culture vessel allows for sensitive evaluation of the neuronal activity of a test substance, and have thus completed the present invention.

[0009] That is, the present invention relates to the following. [1] A method for evaluating the neural activity of a test substance, characterized by comprising the following steps (A) to (E) in order: (A) culturing cultured neurons on the surface of a molded article comprising a norbornene-based polymer, at least the surface of which has been hydrophilized; (B) contacting the cultured neuronal cells with a test substance; (C) fixing the cultured neurons; (D) A step of visualizing drebrin clusters in dendritic spines of the cultured neurons; (E) measuring the linear density of the drebrin clusters along the dendrites, and determining that the test substance has a neuronal effect when the linear density is increased or decreased compared to the linear density of a cultured neuron that has not been contacted with the test substance; [2] The method according to [1] above, wherein the norbornene polymer is a hydrogenated ring-opening polymer of a norbornene monomer. [3] The method according to [1] or [2] above, characterized in that the following step (F) is provided after step (B). (F) contacting the cultured neuronal cells with a glutamate solution; [4] The method according to any one of [1] to [3] above, wherein the cultured neurons are derived from hippocampal neurons. [5] The method according to any one of [1] to [4] above, wherein the cultured nerve cells are derived from a rodent. [6] The method according to any one of [1] to [5] above, wherein the cultured neurons are cultured Alzheimer's disease model neurons. [7] The method according to any one of [1] to [6] above, wherein the visualization in step (D) is performed by immunostaining with an anti-drebrin antibody. [8] A kit for evaluating the neural activity of a test substance, comprising cultured nerve cells and a molded article made of a norbornene-based polymer, at least the surface of which has been hydrophilized. [9] A nerve cell culture vessel for evaluating the neural activity of a test substance, characterized by comprising a molded article made of a norbornene-based polymer, at least the surface of which has been hydrophilized. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for evaluating the neural activity of a test substance that is highly sensitive and has excellent quantitative and reproducible properties, particularly in the low concentration range, as well as a kit and a nerve cell culture vessel for use in the method for evaluating the neural activity of a test substance. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 shows the results of Example 1, comparing a conventional polystyrene 96-well plate with a COP plate. [Figure 2] FIG. 1 shows the results of comparing cultured neurons derived from the hippocampus of a healthy mouse with cultured neurons derived from the hippocampus of an Alzheimer's disease model mouse, using a COP plate, in Example 1. [Figure 3] FIG. 1 shows the results of detecting trace amounts of neurotoxic substances using a COP plate in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method for evaluating the neural activity of a test substance in the present invention is a method (hereinafter sometimes referred to as "the method of the present invention") sequentially comprising the steps of: (A) culturing cultured neurons on the surface of a molded article composed of a norbornene-based polymer, at least the surface of which has been hydrophilized; (B) contacting the cultured neurons with a test substance; (C) fixing the cultured neurons; (D) visualizing drebrin clusters on dendritic spines of the cultured neurons; and (E) measuring the linear density of the drebrin clusters along the dendrites, and determining that the test substance has a neural activity when the linear density increases or decreases compared to the linear density of cultured neurons not contacted with the test substance. Furthermore, the kit for evaluating the neural activity of a test substance in the present invention is a kit for use in evaluating the neural activity of a test substance, comprising cultured neurons and a molded article composed of a norbornene-based polymer whose surface has been hydrophilized (hereinafter, sometimes referred to as the "kit of the present invention"). The neural cell culture vessel for evaluating the neural activity of a test substance in the present invention is a neural cell culture vessel for use in evaluating the neural activity of a test substance, characterized by comprising a molded article composed of a norbornene-based polymer whose surface has been hydrophilized (hereinafter, sometimes referred to as the "neuron culture vessel of the present invention").

[0013] The present invention was completed based on the discovery that, when commercially available hydrophilized polystyrene culture vessels were used, no difference was observed in drebrin-positive synapses and drebrin accumulation (drebrin clusters) after glutamate treatment between hippocampal neurons derived from Alzheimer's disease model mice and hippocampal neurons derived from healthy mice (top panel of Figure 1), whereas when hydrophilized culture vessels made from cycloolefin polymer (COP), a type of norbornene polymer, were used, a difference was detectable, as shown in the bottom panel of Figure 1. Although the reason for this is unclear, it is believed that trace amounts of secreted proteins contribute to the phenotypic expression of cultured neurons derived from the hippocampus of Alzheimer's disease model mice, and that when commercially available hydrophilized polystyrene culture vessels were used, the secreted proteins were nonspecifically adsorbed to the surface of the culture vessel, preventing the expression of the phenotype required for the Alzheimer's disease model. However, by hydrophilizing the cycloolefin polymer (COP) culture vessels, nonspecific adsorption of secreted proteins was suppressed, allowing the difference in phenotype to be detected. Furthermore, culture vessels made of cycloolefin polymer (COP) and subjected to hydrophilization treatment were found to be excellent not only for evaluating extracellular secretory proteins but also for evaluating the neuronal effects of externally added toxic substances. Hereinafter, aspects of the present invention will be described in detail.

[0014] In the present invention, cultured neurons are cultured on the surface of a molded article made of a norbornene-based polymer, at least the surface of which has been hydrophilically treated. The molded article may have a surface on which cultured neurons adhere and are cultured, which surface is made of at least a norbornene-based polymer. Alternatively, the surface of the molded article may be made solely of a norbornene-based polymer, or the entire molded article may be made solely of a norbornene-based polymer. A norbornene-based polymer is a polymer containing monomer units having a norbornene skeleton in an amount of 50% by mass or more, preferably 60% by mass or more, based on the total monomer units constituting the norbornene-based polymer. More specifically, norbornene-based polymers are obtained by polymerizing norbornene-based monomers, which are monomers having a norbornene skeleton, and are roughly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0015] Examples of materials obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers that are ring-opening copolymerizable with the norbornene-based monomers, and hydrogenated products of these. Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith. The norbornene polymers can be used either alone or in combination of two or more. Among these, the hydrogenated ring-opening polymer of a norbornene-based monomer is preferred because the effects of the present invention can be more easily obtained.

[0016] Norbornene monomers that can be used to synthesize norbornene polymers include bicyclo[2.2.1]hept-2-ene (common name: norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2.2.1]hept-2-ene, and 5-methyl-bicyclo[2.2.1]hept-2-ene. ]hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, and other bicyclic monomers; Tricyclo[4.3.0 1,6 .1 2,5 ] Tricyclic monomers such as deca-3,7-diene (trivial name: dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene; Tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyltetracyclo[4.4.0.12,5 .1 7,10 ]-3-dodecene, 8-ethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethyl-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 7,8-benzotricyclo[4.3.0.1 2,5 ] tetracyclic monomers such as dec-3-ene (trivial name: methanotetrahydrofluorene; also known as 1,4-methano-1,4,4a,9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a,9a-tetrahydrofluorene, and 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene; and the like. These norbornene-based monomers may have one or more substituents. Examples of the substituents include alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups. Among these, non-polar substituents such as alkyl groups, alkylene groups, and aryl groups that do not contain atoms with lone electron pairs are preferred. The norbornene polymers can be used either alone or in combination of two or more.

[0017] Other monomers capable of ring-opening copolymerization with norbornene-based monomers include monocyclic cycloolefin-based monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene. These other monomers may have one or more substituents. Examples of the substituents include alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups. Among these, nonpolar substituents such as alkyl groups, alkylene groups, and aryl groups that do not contain atoms with lone electron pairs are preferred. The norbornene polymers can be used either alone or in combination of two or more.

[0018] Other monomers that can be addition copolymerized with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, and tetracyclo[9.2.1.0]. 2,10 .0 3,8 ] Cycloolefin-based monomers such as tetradeca-3,5,7,12-tetraene (also known as 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); non-conjugated diene-based monomers such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene; and the like. Among these, as the other monomer capable of addition copolymerization with the norbornene-based monomer, an α-olefin-based monomer is preferred, and ethylene is more preferred. These other monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group. Among these, non-polar substituents that do not contain an atom having a lone electron pair, such as an alkyl group, an alkylene group, or an aryl group, are preferred.

[0019] A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst composed of a halide of a metal such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst composed of a halide or an acetylacetone compound of a metal such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used. The hydrogenated ring-opening polymer of a norbornene-based monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.

[0020] An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. As the addition polymerization catalyst, for example, a catalyst comprising a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

[0021] Although there are no particular limitations on the molecular weight of the norbornene polymer, the weight-average molecular weight, calculated as polystyrene, measured by gel permeation chromatography (GPC) of a cyclohexane solution (or a toluene solution if the polymer is insoluble) is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. A weight-average molecular weight within this range is preferred because it provides a high level of balance between mechanical strength and moldability.

[0022] The glass transition temperature of the norbornene polymer may be appropriately selected depending on the intended use, but is usually 50 to 300° C., preferably 100 to 280° C., particularly preferably 115 to 250° C., and further preferably 130 to 200° C. When the glass transition temperature is within this range, heat resistance and moldability are well balanced, which is preferable. The glass transition temperature of the norbornene polymer is measured in accordance with JIS K 7121.

[0023] The norbornene polymers can be used either alone or in combination of two or more. Furthermore, in addition to the norbornene-based polymer, the resin component constituting the surface of the molded article may optionally contain additives typically used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent brighteners, in typically employed amounts. When the soft polymer is mixed with the norbornene-based polymer and used, the amount of the soft polymer is typically 0.01 to 20 parts by mass, preferably 0.05 to 10 parts by mass, and more preferably 0.05 to 5 parts by mass, per 100 parts by mass of the alicyclic structure-containing norbornene-based polymer.

[0024] Furthermore, as a resin component constituting the surface of the molded article, in addition to the norbornene-based polymer and the soft polymer, which is one of the compounding ingredients, other polymers (hereinafter simply referred to as "other polymers") may be mixed in. The amount of other polymers mixed with the norbornene-based polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass or less, per 100 parts by mass of the norbornene-based polymer. If the ratio of various additives or other polymers to the norbornene-based polymer is too high, cells will be less likely to float, so it is preferable to add them in an amount that does not impair the properties of the alicyclic structure-containing polymer.

[0025] The method for mixing a norbornene polymer with a compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. The order of compounding is also not particularly limited. Examples of the compounding method include a method of kneading a resin in a molten state using a mixer, a single-screw kneader, a twin-screw kneader, a roll, a Brabender, an extruder, or the like; a method of dissolving and dispersing the resin in a suitable solvent, and then removing the solvent by a coagulation method, a casting method, or a direct drying method; and the like. When a twin-screw kneader is used, after kneading, the material is usually extruded in a molten state into a rod shape, cut to an appropriate length with a strand cutter, and pelletized for use.

[0026] Such a norbornene-based polymer can be generally molded into a molded article having any shape by any molding method, such as injection molding, extrusion molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, or cast molding. An example of a molded article made of a norbornene-based polymer (norbornene-based polymer molded article) is a culture vessel made of a norbornene-based polymer. For example, a dish-shaped culture vessel can be formed by injection molding or the like. It is sufficient that the surface of the culture vessel, to which the cultured nerve cells are attached and cultured, is at least made of a norbornene-based polymer. In the present invention, the surface on which cultured nerve cells are attached and cultured may consist solely of a norbornene-based polymer, or the entire culture vessel may consist solely of a norbornene-based polymer.

[0027] The molded article obtained by molding is subjected to a hydrophilization treatment on its surface before use in the method or kit of the present invention. The hydrophilization treatment allows the article to exhibit adhesive properties suitable for neuronal culture while suppressing nonspecific adsorption of test substances and trace substances secreted by cultured cells, thereby enabling highly sensitive evaluation of the neuronal activity of test substances. Examples of hydrophilization treatment include known hydrophilization methods such as plasma irradiation, UV irradiation, and gamma ray irradiation, which can be applied according to standard procedures. Furthermore, the hydrophilized norbornene polymer molded article may be further coated with a known coating (e.g., poly-L-lysine, poly-D-lysine, collagen, fibronectin, laminin, etc.) to promote adhesion of cultured neurons.

[0028] The norbornene polymer molded article is usually sterilized before use. There are no particular limitations on the sterilization method, and it can be appropriately selected from methods commonly used in the medical field, such as heating methods such as high-pressure steam and dry heat; radiation methods that irradiate with radiation such as gamma rays and electron beams, and irradiation methods that irradiate with high-frequency waves; gas methods that involve contact with gas such as ethylene oxide gas (EOG); and filtration methods that use a sterilizing filter, depending on the shape of the molded body and the cells used.

[0029] The present invention evaluates the neuronal effect of a test substance through its activating and / or inhibiting activity against NMDA-type glutamate receptors, using as an index changes in the linear density of drebrin clusters formed along the dendrites of cultured neurons. Therefore, the cultured neurons in step (A) may be any cells in which drebrin clusters are reduced by NMDA-type glutamate receptor activation. Examples of such cells include primary cultured neurons, hybridomas of neuroblastoma and glioma, neuroblastoma, and cultured neurons derived from pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells). Primary cultured neurons derived from the hippocampus are preferred. Furthermore, the cultured neurons are preferably derived from a source that can be used to evaluate the inhibitory activity of human NMDA-type glutamate receptors. For example, the cultured neurons are derived from mammals, more preferably from primates, and even more preferably from humans. However, cultured neurons derived from rodents such as mice and rats can also be used. Furthermore, the cultured neurons may be cultured neurons derived from human fetuses.

[0030] In the present invention, the neuroactivity of a test substance may be evaluated using cultured neurons derived from healthy animals, or may be evaluated using cultured neurons derived from a pathological model animal such as Alzheimer's disease. Specific examples of embodiments using cultured neurons derived from healthy animals include screening, neuroactivity evaluation, and high-sensitivity detection using candidate compounds for new psychoactive substances (NPSs; also known as "dangerous drugs" in Japan), such as known NPS analogs such as phencyclidine, methoxetamine, and diphenidine, and analogs of NMDA-type glutamate receptor antagonists such as ketamine, as test substances, as well as screening and neuroactivity evaluation using candidate compounds for pharmaceuticals such as anesthetics as test substances. Specific examples of embodiments using cultured neurons derived from a pathological model animal include screening and neuroactivity evaluation using candidate compounds for therapeutic drugs as test substances. Furthermore, test substances are not limited to substances added from external sources, but also include substances secreted by cultured neurons themselves (e.g., autocrine amyloid β). Since the present invention enables highly sensitive evaluation of the neurological effects of test substances, the final concentration of the test substance in step (B) is not particularly limited. 50 and IC 50 When calculating the β-amyloid ratio (β), it is preferable to use a dilution series in which the test substance is serially diluted so as to obtain concentrations that sandwich a 50% activity rate or a 50% inhibition rate. In step (B) above, the contact time of the test substance may be any time sufficient for the test substance to act on the NMDA-type glutamate receptor, and can be, for example, 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 8 to 12 minutes.

[0031] The fixation of cultured neurons in step (C) above may be performed using any known method as long as the conditions allow efficient visualization of drebrin clusters after fixation. Examples of fixation reagents include methanol, acetone, formaldehyde, paraformaldehyde (PFA), ethanol, glutaraldehyde, and dimethyl suberiminate. Fixation reagents that can be used include those cooled in a freezer (-20±2°C) or refrigerator (4±2°C), and those at room temperature.

[0032] In step (D), visualization of drebrin clusters in cultured neurons can be performed by any known method, including, for example, a method using a molecule capable of specifically binding to drebrin. Examples of molecules capable of specifically binding to drebrin include anti-drebrin antibodies. From the standpoints of ease of detection and distinguishability when co-staining with other cultured neuron markers or when staining cells with 4',6-diamidino-2-phenylindole (DAPI), it is preferable to use a fluorescently labeled anti-drebrin antibody. Examples of fluorescent labels include fluorescent substances such as FITC, Cy3, Cy5, Rhodamine, and Alexa Fluor (registered trademark, manufactured by Invitrogen). Anti-drebrin antibodies may be directly labeled or may be detected using a secondary antibody against these. The antibodies used in the present invention may be either monoclonal or polyclonal, and commercially available antibodies may be used, or they may be prepared by conventional methods. Furthermore, cultured neurons may be co-stained with other cultured neuron markers, such as anti-MAP2 antibodies.

[0033] In one embodiment, visualization of drebrin clusters can be performed, for example, by expressing an anti-drebrin antibody (such as an anti-drebrin camel VHH antibody) in cultured neurons and detecting the accumulation of the anti-drebrin antibody. Anti-drebrin antibodies may be detected with a secondary antibody, but real-time imaging using live cells can also be performed by expressing an anti-drebrin antibody bound to a labeling substance such as a fluorescent protein in cultured neurons and detecting the labeling substance. Furthermore, in one embodiment, when drebrin or a drebrin fragment bound to a labeling substance such as a fluorescent protein is expressed in cultured neurons, the expressed labeled drebrin or drebrin fragment forms drebrin clusters. In this embodiment, drebrin clusters can be detected by detecting the labeled drebrin or drebrin fragment without an additional visualization step. In these embodiments, fixation of the cultured neurons in step (C) may or may not be performed.

[0034] The linear density of drebrin clusters in step (E) is measured by manually or automatically counting the number of drebrin-positive signals visualized in step (D) and calculating the number of drebrin-positive signals per unit length of the dendrite. Measurement of such linear density is preferably performed automatically using known image analysis software such as IN Cell Developer Toolbox (GE Healthcare).

[0035] The method of the present invention may further comprise, after step (B), step (F) of contacting the cultured neurons with a glutamate solution. Here, the glutamate solution activates NMDA-type glutamate receptors, resulting in a decrease in drebrin clusters. Therefore, in a method comprising step (F), if the addition of a test substance suppresses the decrease in drebrin clusters, it can be determined that the test substance has an inhibitory effect on the formation or function of NMDA-type glutamate receptors. The final concentration and contact time of the glutamate solution may be selected so as to induce NMDA-type glutamate receptor activation appropriate for evaluating the NMDA-type glutamate receptor inhibitory effect of the test substance. Examples of the concentration include 1 μM to 500 μM, preferably 10 μM to 300 μM, more preferably 50 μM to 200 μM, and even more preferably 80 μM to 150 μM. Examples of the contact time include 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 8 to 12 minutes. Furthermore, instead of the glutamate solution, any compound capable of inducing NMDA-type glutamate receptor activation may be used.

[0036] The kit of the present invention and the nerve cell culture vessel of the present invention are preferably suitable for carrying out the method of the present invention, and can have the configurations disclosed in this specification regarding the carrying out of the method of the present invention.

[0037] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0038] 1. Comparison of changes in drebrin cluster density by glutamate treatment between healthy mice and Alzheimer's disease model mice 1-1 Method Cultured neurons prepared from the hippocampus of embryonic day 16 normal mice and Alzheimer's disease model mice were plated at 4.5 × 10 cells / well onto poly-L-lysine-coated 96-well polystyrene plates (CELLSTAR, Advanced TC, product number 655986, GreinerBio-One International) and plasma-treated hydrogenated norbornene ring-opening polymer (ZEONOR® 1060R, Zeon Corporation) 96-well plates (hereinafter also referred to as COP plates). 4 cells / cm 2 The cells were seeded at a density of 1000 μg / well. They were incubated at 37°C in a 5% CO2 environment using Neurobasal Medium (Thermo Fisher Scientific) containing B27 supplement, penicillin / streptomycin, and L-alanyl-L-glutamine (Glutamax-I; Thermo Fisher Scientific). After 21 days of in vitro culture, the cells were treated with glutamate solution (Wako Pure Chemical Industries, Ltd.) at final concentrations of 0 μM, 1 μM, 10 μM, 30 μM, or 100 μM for 10 minutes.

[0039] Cultured neurons were then fixed with 4% paraformaldehyde in phosphate buffer. After permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 minutes, the cultured neurons were blocked with 3% bovine serum albumin in PBS (PBSA) for 1 hour at room temperature (RT) and then incubated with primary antibodies overnight at 4°C. Anti-drebrin antibody (mouse monoclonal, M2F6 hybridoma cell culture supernatant, 1:1) and anti-MAP2 antibody (rabbit polyclonal, 1:2000, Merck Millipore) were used as primary antibodies. After washing with PBS, the cells were incubated with secondary antibodies and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:1000, Thermo Fisher Scientific) for 1 hour at room temperature. The secondary antibodies used were Alexa Fluor 488 donkey anti-mouse IgG (1:250) and Alexa Fluor 594 donkey anti-rabbit IgG (1:250, Jackson Immuno Research Laboratories).

[0040] Triple-stained images of hippocampal neurons cultured in 96-well plates were automatically acquired using the autofocus function of an IN CellAnalyzer 2200 (GE Healthcare) (20x lens, 0.45 numerical aperture). All data were collected at a resolution of 2048 × 2048 pixels with 16 bits per pixel. A single pixel in the image corresponded to a 325 nm square in the specimen plane. Z-stack images were processed using the high-content imaging software IN CellDeveloper Toolbox v1.9 (GE Healthcare) with an extended focus transformation protocol to generate a single, in-focus, two-dimensional image from the Z-series stack. Cultured neuronal cell bodies were identified based on MAP2 and DAPI staining. To identify cultured neuronal cell bodies, the segmentation function localized fluorophores based on the fluorescence intensity of the MAP2-positive signal via a contrast-based segmentation algorithm with different kernel sizes and sensitivities. The Erosion, Sieve, and Dilation commands and the DAPI-positive area were used to identify the cell bodies of cultured neurons. To identify the dendrites of cultured neurons, the Segmentation function and the Dilation and Erosion commands were used with separate parameters for the MAP2-positive signal, after which the cell body area of ​​the cultured neurons was subtracted. To identify drebrin clusters, the Segmentation function localized fluorescent objects based on the fluorescence intensity of the drebrin-positive signal through a contrast-based segmentation algorithm with different kernel sizes and sensitivities. The Sieve command was used to remove false-positive background signals. In addition, only drebrin-positive puncta located within a defined distance to the dendrite, as determined by the Dilation command, were counted as drebrin clusters along the dendrites. After identifying the neuronal cell bodies, dendrites, and drebrin clusters, the number of cells per field, and the dendritic length per field, the linear density of drebrin clusters was automatically calculated.The test for significant difference was performed using Dunnett's method following ANOVA (Excel Statistics Statcel 4, OMS Publishing Co., Ltd.).

[0041] 1-2 Results Figure 1 shows the results of comparing a conventional polystyrene 96-well plate with a COP plate. The linear density of drebrin clusters was measured in three wells per glutamate concentration, and the change in drebrin cluster density was calculated by setting the density of wells to which glutamate had not been added as 1. The significant difference from wells to which glutamate had not been added was then examined. When the conventional polystyrene 96-well plate (shown as "greiner" in the upper panel of Figure 1) was used, a significant decrease in drebrin clusters was observed at a glutamate concentration of 30 μM in both cultured neurons derived from the hippocampus of a healthy mouse (WT on the left) and cultured neurons derived from the hippocampus of an Alzheimer's disease model mouse (AD on the right), and contrary to expectations, no difference in NMDA-type glutamate receptor activity was detected. On the other hand, when the COP plate of the present invention was used (lower panel of Figure 1), no glutamate concentration-dependent decrease in drebrin clusters was observed in cultured neurons derived from the hippocampus of an Alzheimer's disease model mouse, and a decrease in NMDA-type glutamate receptor activity in the Alzheimer's disease model mouse was detected.

[0042] Figure 2 shows the results of comparing cultured neurons derived from the hippocampus of healthy mice with those derived from the hippocampus of Alzheimer's disease model mice using a COP plate. The linear density of drebrin clusters was measured in six wells per glutamate concentration, and the actual values ​​and the significant differences relative to wells without glutamate were plotted. These results demonstrate that, as in Figure 1, cultured neurons derived from the hippocampus of healthy mice (WT, left) exhibited a significant decrease in drebrin clusters at a glutamate concentration of 30 μM. Cultured neurons derived from the hippocampus of Alzheimer's disease model mice (AD, right) consistently exhibited lower linear density of drebrin clusters than wells without glutamate. These results suggest that trace amounts of secreted proteins, such as amyloid beta, may contribute to the phenotypic expression of cultured neurons derived from the hippocampus of Alzheimer's disease model mice. The COP plate was able to detect the Alzheimer's disease model phenotype, which was not detectable in a hydrophilic-treated polystyrene 96-well plate. The reason for this is presumably that the nonspecific adsorption of secreted proteins was suppressed in the case of the COP plate, making it possible to detect the phenotype as an Alzheimer's disease model. [Example]

[0043] 2. Detection of trace neurotoxic substances using COP plates Example 1 suggests that plasma-treated COP plates suppress nonspecific adsorption of extracellular secretory proteins to the plate surface, and that even trace amounts can have an effect. Therefore, we investigated whether plasma-treated COP plates can detect even trace amounts of exogenous cytotoxic substances.

[0044] 2-1 Method Cultured neurons prepared from the hippocampus of 18-day-old embryonic rats were plated at 3.0 × 10 cells per well onto a poly-L-lysine-coated 96-well polystyrene plate (CELLSTAR, Advanced TC, product number 655986, GreinerBio-One International) and a 96-well plate (hereinafter also referred to as COP plate) made of hydrogenated norbornene ring-opening polymer (ZEONOR® 1060R, Zeon Corporation) plasma-treated by a conventional method. 4 cells / cm 2 The cells were seeded at a density of 100 μg / ml. They were incubated at 37°C in a 5% CO2 environment using Neurobasal Medium (Thermo Fisher Scientific) containing B27 supplement, penicillin / streptomycin, and L-alanyl-L-glutamine (Glutamax-I; Thermo Fisher Scientific). At 4 days in vitro (DIV), cytosine β-D-arabinofuranoside (Sigma) was added to a final concentration of 0.2 μM to inhibit glial cell proliferation. At DIV 10, purified amyloid β (0, 100, or 600 nM) was added, and at DIV 22, the cells were fixed with 4% paraformaldehyde in phosphate buffer. From this point onward, the linear density of drebrin clusters was measured as in Example 1. The linear density of drebrin clusters in 6 wells per purified amyloid β concentration was measured, and significant differences were tested using GraphPad Prism8 (MDF Co., Ltd.) by performing two-way ANOVA followed by Dunnett's multi-comparisons test.

[0045] 2-2 Results The results are shown in Figure 3. In the COP plate, a concentration-dependent decrease in drebrin cluster density was observed following administration of amyloid beta for 12 days from DIV10 to DIV22, but this phenomenon was not detected in a polystyrene 96-well plate coated with poly-L-lysine (shown as Greiner in Figure 3). Therefore, it became clear that the COP plate is also excellent for detecting trace amounts of exogenous cytotoxic substances. [Industrial Applicability]

[0046] The present invention provides a method for evaluating the neuronal activity of a test substance that is highly sensitive and has excellent quantitation and reproducibility, particularly in the low concentration range, and can be applied to, for example, the following: 1. The present invention makes it possible to detect the chronic neurotoxicity of trace amounts of secreted amyloid beta by a phenotypic assay using cultured neurons, thereby facilitating drug development using neurons derived from Alzheimer's disease model mice and neurons derived from iPS cells of Alzheimer's disease patients. 2. It is now possible to measure the activity of minute amounts of physiologically active substances, which was difficult to measure quantitatively using conventional assays using cultured adherent cells. 3. Because there is little nonspecific adsorption of physiologically active substances and drugs to the culture vessel, improved quantitation and reproducibility (especially in the low concentration range) can be expected. 4. A toxicity test method is provided which includes a step of culturing adherent disease model cells (which may be genome-edited or derived from a diseased organism) in a culture vessel made of cycloolefin polymer. Therefore, the present invention has extremely high industrial applicability in the medical and pharmaceutical fields.

Claims

1. A method for evaluating the neural activity of a test substance, comprising the following steps (A), (B), (F), and (C) to (E) in that order: (A) culturing hippocampal-derived Alzheimer's disease model cultured neurons on the surface of a molded article comprising a norbornene-based polymer, at least the surface of which has been hydrophilized; (B) contacting the hippocampus-derived Alzheimer's disease model cultured neurons with a test substance; (F) contacting the hippocampus-derived Alzheimer's disease model cultured neurons with a glutamic acid solution; (C) fixing the hippocampus-derived Alzheimer's disease model cultured neurons; (D) visualizing drebrin clusters in dendritic spines of the hippocampus-derived cultured Alzheimer's disease model neurons; (E) measuring the linear density of the drebrin clusters along the dendrites, and determining that the test substance has a neuronal effect when the linear density is increased or decreased compared to the linear density of a cultured neuron that has not been contacted with the test substance;

2. 2. The method according to claim 1, wherein the norbornene polymer is a hydrogenated ring-opening polymer of a norbornene monomer.

3. The method described in claim 1 or 2, characterized in that the hippocampus-derived Alzheimer's disease model cultured neurons are derived from rodents.

4. The method according to any one of claims 1 to 3, wherein the visualization in step (D) is performed by immunostaining with an anti-drebrin antibody.

5. A kit for use in the method described in any one of claims 1 to 4, characterized in that it comprises hippocampal-derived Alzheimer's disease model cultured neurons, a molded body composed of a norbornene-based polymer with at least the surface hydrophilized, and a glutamic acid solution.

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