WO2022154080A1 - In vitro neural circuit tissue exhibiting complex activity and plasticity, and device for observing said neural activity and method for screening substances using said device - Google Patents

Abstract

The present invention provides a neural circuit tissue that is induced in vitro and that has at least two nerve cell masses (e.g., organoids) connected via axons. The present invention also provides a device for observing the neural activity of this neural circuit tissue and a method for screening substances using this device. This neural circuit tissue carries out spontaneous firing activity and is characterized in that two nerve cell masses exhibit interrelated activity, and in particular can be used as a brain model.

Description

In vitro neural circuit tissue showing complex activity and plasticity, an observation device for the neural activity, and a method for screening substances using this

The present invention relates to an in vitro induced neural circuit tissue exhibiting complex activity and plasticity, an observation device thereof, and a method for screening a substance that changes the neural activity of the neural circuit tissue using the observation device.

Many areas in the brain are connected, and they produce advanced functions by interacting with each other and influencing them. It is thought that understanding the connections between regions can clarify the mechanism by which the brain functions, but it is easy to analyze because the complexity of neural circuits in the brain is a major barrier to functional analysis of the brain. , A simplified in vitro neural circuit model is needed.

In recent years, attempts have been actively made to understand the development and diseases of the brain using brain organoids produced in vitro. The organoid production technique is a technique for three-dimensionally modeling an animal organ in vitro, and can be used for functional analysis of various organs in a state closer to in vivo. So far, brain organoids including the cerebral cortex, thalamus, cerebellum, hippocampus and choroid plexus have been reported (see Non-Patent Documents 1 to 6 and the like), and a new platform for studying human brain regions in vitro has been reported. It is being offered.
However, although the structural and morphological characteristics of brain organoids reported so far are similar to those of the human brain at the developmental stage, it is considered that functional improvements are required. There is. Since it is considered to be the basis of higher-order functions, a brain organoid model tissue having the property of exchanging activities between regions and having plasticity as a neural circuit is particularly required.
Further, in order to obtain an organoid showing a complicated activity pattern, long-term (3 months or more) culture is required, and a method for obtaining an organoid showing a complicated activity in a short culture period is required.

As an in vitro model of nerve tissue, brain organoids connected by axon bundles have been reported (Non-Patent Document 7 and Patent Document 1). This "connected organoids" model is constructed by connecting two brain organoids via a bundle of axons extending from each organoid. This model reflects the state of the in vivo brain in which brain regions are connected by axon bundles, and is considered to be a very useful model in brain research.
On the other hand, "fused organoids" technology has also been developed in which two or more brain organoids are directly fused by adjoining them (Non-Patent Document 8 and the like).
However, it was unclear what kind of activity and function these in vitro neural circuit models exert.

WO2017 / 187696 Gazette

Del Dosso et al., Neuron 107 1014-1028 2020. Xiang et al., Cell Stem Cell 21 383-398 e7 2017. Sakaguchi et al., Nat Communi. 6 8896 2015. Muguruma et al., Cell Rep. 10 537-550 2015. Xiang et al., Cell stem cell 24 487-497 e7 2019. Pellegrini et al., Science 369 eaaz5626 2020. Kirihara et al., IScience 14 301-311 2019. Birey et al., Nature 545 54-59 2017.

In view of the above circumstances, it is an object of the present invention to obtain an in vitro induced neural circuit tissue capable of exchanging neural activity between regions and exhibiting complex activity and plasticity.
Another object of the present invention is to provide an observation device for neural activity of neural circuit tissues.
Furthermore, it is an object of the present invention to provide a method for screening a substance that changes the neural activity of the neural circuit tissue by using the neural network tissue induced in vitro and this observation device.

The present inventors cultivate a plurality of brain organoids induced to differentiate from human iPS cells on a multi-electrode array (chip having a plurality of electrodes on the array; MEA), and the brain organoids are connected by an axon bundle. The nerve tissue was prepared and the neuronal activity occurring in the nerve tissue was analyzed. For the first time, we succeeded in detecting extremely complex neuronal activity from organoids connected by axon bundles (hereinafter also referred to as "connected (brain) organoids").

That is, the neuronal activity of the connecting brain organoid was detected as spontaneous firing activity after a relatively short period of culture (about 7-8 weeks after the iPS cell culture). The connected organoids performed burst activity, and the coefficient of variation of the frequency of burst activity was 0.2 or more. In addition, the local field potential (LFP) detected from the nerve tissue is characterized by the δ wavelength band component (0.5 Hz-4 Hz) and / or the θ wavelength body component (4.0 Hz-8.0 Hz) element. It was something that could be done.

Furthermore, it was clarified that when the neuronal activity propagating the axon bundle of the above-mentioned connecting organoid is suppressed by the optogenetic method, the neuronal activity of each organoid is significantly suppressed. This result suggests that axonal bundle connections between organoids are very important in producing active and complex neuronal activity. Furthermore, it was found that stimulation by irradiating the axon bundle of the connecting organoid with light induces neuronal activity in the organoid in response to the stimulus. This neuronal activity was maintained even after the photostimulation was discontinued. In order for the neuron activity in response to such a light stimulus to act in response to the stimulus pattern, a delay period (time lag) was required between the stimulus and the activity. However, it was found that repeated light stimulation shortened this time lag and reproduced the previously performed activity pattern in a short time after the re-stimulation. This suggests that connecting organoids are capable of retaining short-term memory of time information.

The present invention has been completed based on the above findings.
That is, the features of the present invention are listed as (1) to (24) below.
(1) Neural circuit tissue induced in vitro, in which two or more nerve cell clusters are connected via axons (also referred to as "connecting organoids"). The nerve cell mass may be an organoid.
(2) The two or more nerve cell clusters exhibit interrelated activities. The interrelated activities can occur with a time lag of less than 500 milliseconds. Also, the interrelated activities can be synchronous activities.
(3) Spontaneous ignition activity is performed, and the spontaneous ignition activity may be 50 times or more per minute.
(4) Burst activity is performed, and the coefficient of variation of the frequency of the burst activity can be 0.2 or more.
(5) The activity of each nerve cell mass connected via the axon exhibits coherence, and the interference may differ from frequency band to frequency band.
(6) The local field potential detected from the nerve cell mass includes a δ wavelength band (0.5 Hz-4.0 HZ) component and / or a γ wavelength band (300 Hz-3000 Hz) component. Further, the local field potential may further include a θ wavelength band (4.0 Hz-8.0 Hz) component.
(7) The nerve cell mass may be one in which differentiation is induced from pluripotent stem cells, and the nerve cell mass may be formed by culturing pluripotent stem cells for 6 weeks or more. Further, the pluripotent stem cell can be an iPS cell (induced pluripotent stem cell).
(7) It is obtained by culturing the nerve cell mass for 2 weeks or more.
(8) Shows plasticity against external stimuli.
(9) An in vitro induced neural activity observation device in which two or more nerve cell masses are connected via an axon, and is a substrate and the substrate for accommodating the nerve cell mass. A plurality of wells provided on the surface of the above, and an induction groove provided by connecting the wells for inducing and extending the axons so as to connect the nerve cell masses to each other. Each well is provided with an electrode and further includes an analysis unit that analyzes the correlation of electrical signals from the electrode.
(10) The analysis unit separates each of the electric signals for each frequency band and analyzes the correlation by phase amplitude coupling.
(11) The analysis unit analyzes by wavelet coherence.
(12) The analysis unit classifies the neural activity into any of action potential, burst activity, brain hypothesis, and local field potential.
(13) The analysis unit has a signal pattern of the electric signal corresponding to the nerve activity in advance, and classifies the nerve activity by collating with the signal pattern.
(14) The nerve cell mass and / or the axon can be externally stimulated to form the nerve activity, and the obtained signal pattern can be given in advance.
(15) The stimulus can be any one or more of light irradiation, electrical stimulation or compound administration. Also, the stimulus can enhance or suppress the neural activity.
(16) The electrode is formed by arranging a plurality of electrodes in an array at the bottom of the well.
(17) The electrode receives the electric signal from the nerve cell mass and electrically stimulates the nerve cell mass.
(18) The substrate is transparent so that the nerve cell mass in the well can be optically observed from the bottom.
(19) A light irradiation device for optically stimulating the nerve cell mass and / or the axon is provided so as to face the surface of the substrate.
(20) The light irradiation device includes an irradiation unit that locally applies light irradiation to the nerve cell mass and / or a part of the axon.
(21) An in vitro induced neural activity observation device in which two or more nerve cell masses are connected via an axon, the substrate and the above-mentioned device for accommodating the nerve cell mass. The wells include a plurality of wells provided on the surface of the substrate and a guide groove provided by connecting the wells for guiding and extending the axons so as to connect the nerve cell clusters. Each of the above is provided with an optical system for measuring the light intensity from the nerve cell mass, and further includes an analysis unit for acquiring the correlation of the light intensity signal from each of the nerve cell clusters in the well. And.
(22) Two wells are provided to the substrate, the first objective lens of the optical system is brought close to one of the wells from one surface of the substrate, and the second objective lens is the other of the substrate. It can be provided close to the other side of the well from the surface of the above.
(23) The emission from the calcium fluorescent probe can be detected, and the increase in calcium ion concentration in the nerve cell mass caused by the action potential can be detected. Here, the light beam for causing the light emission can be incident from the side end surface of the substrate.
(24) A method of screening a substance that changes the neural activity of a neural circuit tissue using an observation device having the above-mentioned characteristics. The target neural circuit tissue is set in the observation device, and the substance is used as the nerve. It is characterized in that it is given to a circuit tissue and the fluctuation of the neural activity is observed.
In addition, in this specification, the symbol of "-" indicates a numerical range including the values to the left and right of the reference numeral.

The neural circuit tissue according to the present invention mimics the function of the brain in vitro, and by combining with the observation device according to the present invention, various useful information can be obtained by observing (analyzing) this. For example, it is possible to screen for substances that may fluctuate the neural activity of the brain by using the neural circuit tissue and the observation device, which is one of the neural circuit tissues according to the present invention and is composed of cerebral organoids. Such findings are useful for searching for therapeutic agents for psychiatric disorders and neurodegenerative diseases as a brain disease model including disorders of higher brain function.

Formation and characterization of connected organoids on PDMS-MEA chips. Analysis of neuronal activity of brain organoids connected by axon bundles. Genetic analysis of linked brain organoids (connecting organoids) by Single cell RNA seq. Analysis of axon bundle characteristics using photoconverted fluorescent protein. Examination of optogenetic inhibition of burst activity between connected organoids and synchronization between both organoids. Results of analysis of complex activities occurring in connected organoids using Phase-Amplitude Coupling and hidden Markov models. Analysis of short-term memory mechanisms in connected organoids. Analysis of various burst activity patterns supported by CaMKII-dependent signals. Visualization of elongated axons of connecting organoids. Comparison of neural activity of single organoids, fused organoids and connected organoids. Measurement results of axon conduction velocity of organoids connected by axon bundles of various lengths. Analysis results of neuronal activity in organoids in which axon bundles are physically cleaved. Results of investigating changes in LFP associated with maturation of connected organoids and the effect of drug treatment on neural activity of connected organoids. Scalogram of LFP signal and wavelet transform of connecting organoids in the presence of various agents. Simultaneous measurement results of Ca ²⁺ transients and electrical activity.

Hereinafter, embodiments of the present invention will be described.
The first embodiment of the present invention is a neural circuit tissue induced in vitro, in which two or more nerve cell clusters are connected via an axon, a neural circuit tissue (neural circuit according to the present embodiment). Organization).
In the present embodiment, the "cell mass" is a cell population in which cells adhere to each other to form a three-dimensional structure and have a structure similar to the state of existence in a living body. A nerve cell mass is a cell mass composed of nerve cells. An "organoid" is a complex cell cluster consisting of cells derived from or organ-specific cells such as the brain, stomach, liver, and bladder. Organoids can be produced by self-aggregating pluripotent stem cells and the like (see, for example, Non-Patent Documents 1 to 7). As used herein, the term "organoid" means a brain organoid, that is, an organoid containing nerve cells, unless otherwise specified.

The neural circuit tissue according to the present embodiment has a minimum unit of a tissue in which nerve cell clusters such as two brain organoids are connected to each other by a bundle of axons, and three or more nerve cell clusters are mutually axon bundles. It may be connected with. Here, the "neural circuit tissue" is a nerve tissue in which neurons are connected to each other via an axon, and the activity of nerve cells in one brain organoid or nerve cells as a group is the activity of another brain. It is an organization that can interfere with the activity of organoids. This can be defined, for example, by the spontaneous firing activity of each cerebral organoid, the synchrony or asynchrony of neural activity between two or more cerebral organoids, the strength of PAC, and the like. It is a tissue that can perform burst activity and periodic oscillation activity as a result of the coordination of neural activities of multiple organoids. Connected organoids can cause a continuous brain hypothesis. In addition, the cell masses connected to an external stimulus (for example, light irradiation) show a response related to each other and are characterized by having plasticity.
The neural circuit tissue according to the present embodiment is prepared, for example, by placing a nerve cell mass such as one organoid in a small hole (well) connected by a guide (thin groove) for extending an axon and culturing it. can do. Organoids can be produced by culturing pluripotent stem cells such as iPS (induced pluripotent stem cells) cells for a period of about 4 to 6 weeks under appropriate culture conditions (of the nerve tissue according to this embodiment). For details of the production method, refer to Non-Patent Document 7 or Examples).

In the nerve tissue prepared as described above, although the situation differs slightly depending on the culture conditions, the nerve cell mass becomes "spontaneous firing activity" (from the outside world) 1.5 weeks or 2 weeks after the start of the culture of the nerve cell mass. Even if there is no stimulus, a phenomenon that causes spikes with a short time width) will occur. In the neural circuit tissue according to the present embodiment, the number of spontaneous firing activities is, for example, 50 times or more per minute.
Here, the "activity" is a change in a nerve cell caused by an action potential generated in the nerve tissue. Starting from a change in the membrane potential that occurs in the cell membrane due to synaptic activity, the diffusion of sodium ions and potassium ions on the cell membrane by voltage-gated ion channels due to the difference in concentration inside and outside the cell is generally called an action potential.
Although the membrane potential cannot be measured directly by MEA, it is possible to measure the weak change in electrical signal caused by the action potential outside the cell near the nerve cell (extracellular recording). In calcium imaging, the increase in intracellular calcium ion concentration (Ca ²⁺ ) caused by action potential can be observed by detecting the luminescence from the calcium fluorescent probe with a microscope or the like. Changes caused by nerve action potentials recorded by techniques such as those given in these examples are referred to as "activity" of nerve cells and neural circuit tissues.

Furthermore, in addition to the spontaneous firing activity, the neural circuit tissue according to the present embodiment causes a "burst activity" in which a plurality of action potentials are collectively fired at a high frequency. Burst activity can be triggered by the continuous activity of one nerve cell. It can also be caused by the continuous activity of multiple nerve cells. During burst activity, the frequency of observed activity is significantly higher than during non-burst activity.

The characteristic of burst activity in the nervous tissue according to this embodiment is that the coefficient of variation of the frequency of burst activity is, for example, 0.2 or more. Here, the coefficient of variation is a value obtained by dividing the standard deviation of the burst frequency by the average. A coefficient of variation of burst activity of 0.2 or more is an indicator that the activity of neural circuit tissue has "complexity".

Local field potential (LFP) will be detected from the nerve cell mass 1.5 weeks or 2 weeks after the start of culture of the nerve cell mass. This local field potential includes a δ wavelength band (0.5 Hz-4.0 HZ) component, a γ wavelength band (300 Hz-3000 Hz) component, and / or a θ wavelength band (4.0 Hz-8.0 Hz) component.

The neural cell masses in the neural circuit tissue according to this embodiment show interrelated activities.
"Interrelated activity" refers to the neural activity in each neural mass, where any association is found between the connected neural masses, the neural activity in each, or the neuronal activity. Taking the neural circuit tissue in which organoid 1 and organoid 2 are connected as an example, when activity or burst activity occurs in organoid 1, when activity or burst activity occurs in organoid 2 at about the same time, that is, organoid 1 It can be said that the neural circuit tissue has performed interrelated activities when the neural activities in the organoid 2 and the organoid 2 are synchronized. Or, in addition to synchronized activity, when neural activity in one organoid occurs, with a certain time difference, when the other causes the same neural activity, or when the neural activity is mutually timed or with a certain phase difference. It can be said that they carried out activities related to each other even when they were repeated.
Further, as the "interrelated activity", it is desirable that the activity performed by each nerve cell mass of the two nerve cell masses occurs simultaneously or at short time intervals. Although not particularly limited, for example, the activity (related activity) of each nerve cell mass is an activity performed within 1,000 milliseconds, preferably within 500 milliseconds, and more preferably within 100 milliseconds.
Showing interrelated activities with time or phase differences within an organization is also an indicator of "complexity."

In the neural circuit tissue according to the present embodiment, the activities performed by each nerve cell mass connected by the axon bundle may interfere with each other (coherence), and this interference is one of the characteristics of the neural circuit tissue. .. This interference may vary from frequency band to activity pattern. This interference for each frequency band is measured as a degree of linear relevance at each frequency by performing wavelet coherence analysis based on the time series data of the two active waves detected from each nerve cell mass. Can be represented. The wavelet coherence analysis has been described in detail in the Examples section, so please refer to it.
It is also an index of "complexity" that different sites in the neural circuit tissue show wavelet coherence.

Furthermore, the neural circuit tissue according to the present embodiment is characterized by having plasticity against external stimuli (for example, light irradiation). Here, "plasticity" refers to the property of responding to an external stimulus and changing the activity pattern after the stimulus. The neural circuit tissue is characterized by having plasticity.

In the neural circuit tissue according to the present embodiment, means for detecting the neuron activity detected from the neural network tissue may be arranged.
Here, the "means" includes electrodes as the detection means when the neuron activity is detected as an electric signal. The electrical signal of neuron activity is analyzed, for example, by arranging a nerve cell mass on a substrate on which one or more electrodes are arranged and detecting the electrical signal of the activity wave derived from each nerve cell mass from the electrode. be able to. In order to connect a plurality of nerve cell masses to each other with an axon bundle, the nerve cell masses are cultured on a substrate provided with a multi-electrode array, and at the same time, an electrical signal obtained from the nerve cell masses is detected. May be good. Specifically, in the neural circuit tissue according to the present embodiment, small holes (small holes for inserting the nerve cell mass and the medium) capable of culturing the nerve cell mass are arranged on each array of the multi-electrode array, and each of them. Culturing was performed between the small holes using a device in which a groove was arranged so as to serve as an extension guide (also a flow path for the medium) of the axon extending from the nerve cell mass toward another nerve cell mass. You may. The activity of the nerve tissue according to the present embodiment is to manipulate the activity of the nerve tissue (modify the activity of the nerve tissue) by irradiating a part of the nerve tissue (for example, an axon bundle) with light. can. When performing such an operation, the device is provided with an element for giving an external stimulus such as light irradiation, for example, a groove (also serving as a flow path for a medium) for arranging an optical fiber or the like. May be. See FIGS. 1A and 1B, which will be described later, as an example of the device.

Further, for example, as shown in FIG. 15A described later, as a "means" for detecting neuronal activity by a calcium imaging method, an optical system (microscope or the like) for detecting light emission emitted from each nerve cell mass is used. Can be mentioned. For the optical system, a CCD camera or the like that measures the light intensity is used, and the presence or absence of correlation of the light intensity signals from each of the nerve cell clusters is included and analyzed. Here, the first objective lens (Objective 1) of the optical system is brought close to one side of the nerve cell mass from one surface of the substrate, and the second objective lens (Objective 2) is moved from the other surface of the substrate to the nerve cell. The light intensity is measured by bringing it close to the other side of the mass. In this case, in order to detect a change in the calcium ion concentration (Ca ²⁺ ) in the neuron cell, it is advisable to introduce a fluorescent calcium probe into the cell in advance. Examples of proteinaceous fluorescent calcium probes include chameleon, GCaMP (G-CaMP2, G-CaMP4, G-CaMP6, G-CaMP7 and G-CaMP8, etc.), R-CaMP (R-CaMP1.07). , R-CaMP2, etc.). Although not shown in FIG. 15, the light beam for causing fluorescence emission may be incident from the side end surface of the substrate using a transparent substrate.

The second embodiment is a method of analyzing neural activity in vitro, in which the activity of each frequency band obtained from the neural cell mass constituting the neural circuit tissue according to the first embodiment is detected and the presence or absence of correlation is present. It is a method including analyzing the detected activity correlation by a method for analyzing the correlation of neural activity such as Phase-Amplitude-Coupring (PAC) and wavelet coherence.
Here, the phase amplitude coupling was established to calculate a value that is an index of the relationship between the phase of low frequency activity and the amplitude of high frequency spikes, and to evaluate the relationship between brain waves in different frequency bands. The method (Fell et al., Nat. Rev. Neurosci. 12 105-118 2011: Canolty et al., Trends Cogn. Sci. 14 506-515 2010). For example, a delta-gamma (amplitude) PAC can analyze whether the frequency amplitude intensity in the gamma wave region affects the phase in the delta wave region.
According to the method according to the second embodiment, for example, a delta (phase) -gamma (amplitude) PAC or ata (phase) -gamma (amplitude) PAC correlation rate (Modulation index) is associated with the cultivation of brain organoids. Elevation can explain the maturity of brain organoids in terms of neural activity.
The PAC correlation rate of activities in different frequency bands is preferably 0.05 or higher, which is also an indicator of the "complexity" of neural network tissue.

A third embodiment is a method of modulating the neural activity of the neural circuit tissue according to the present embodiment, in which the neural activity of the neural network tissue is recorded and the neural network tissue is stimulated by an arbitrary pattern. It is a method including that.
Here, the "nerve activity pattern" is a spatiotemporal feature or relationship of the activity of a neural circuit tissue or a nerve cell. The neural activity pattern includes, for example, the frequency of "burst activity" (or "burst-like activity"). "Burst activity" means that high-frequency neural activity is concentrated in time during a short period of time (for example, 100 ms) (nerve activity is observed at a significantly higher frequency than during non-burst). It is a characteristic neural activity pattern. Not only the frequency is constant, but also the non-constant frequency (the coefficient of variation of the interval between burst activities is small or large) can be said to be a neural activity pattern. Similar to burst activity, neural activity in a specific frequency band and its correlation can be said to be a neural activity pattern. Not only temporal features and correlations, but also the association of neural activity between electrodes (or between observed data of spatially different sites or positions) is captured as part of the neural activity pattern. Therefore, a neurological avalanche is also one of the neural activity patterns. In addition, spatiotemporal correlation of neural activity between connected neural tissues can also constitute a neural activity pattern.
Further, "stimulating the neural circuit tissue by an arbitrary pattern" means applying stimulation to the neural activity at an arbitrary frequency and place determined spatiotemporally in order to induce neural activity. For example, stimulating at any frequency or stimulating any site. It is also possible to stimulate with a random spatiotemporal pattern. The pattern can be determined in advance as desired, but it can also be calculated and determined based on the observed neural activity pattern.
The "stimulation" is not particularly limited, but is, for example, a stimulus given by light, electricity, compound administration, or the like. The neural tissue circuit according to the present embodiment is characterized in that its activity is enhanced or suppressed by these stimuli.

The fourth embodiment is a method for screening a substance that may fluctuate the neural activity of the brain, in which the candidate substance is brought into contact with the neural circuit tissue according to the present embodiment to detect the neural activity of the neural tissue. It is a screening method including that.
The neural circuit tissue according to this embodiment is spontaneously firing and exhibits an activity pattern similar to that of neural activity in the brain in vivo. In addition, when an external stimulus is repeatedly applied to the nerve tissue (for example, the axon bundle is stimulated by light irradiation), the time to respond to the stimulus becomes shorter as the number of stimuli is increased. In addition, when the axon bundle of the nerve tissue is light-stimulated for a certain period of time (for example, about 20 minutes), the nerve tissue performs burst-like activity, but this burst-like activity disappears immediately after the stimulation is stopped. It has also been clarified that the activity frequency continues to increase for a while. Therefore, it can be understood that the neural circuit tissue according to the present embodiment has a function of storing (remembering) an external stimulus pattern in the tissue. Since the activity supporting the memory in the nerve tissue is suppressed by the CaM kinase II inhibitor, at least the neural circuit tissue according to the first embodiment is the nerve activity of the brain generated during short-term memory. It was suggested that similar activities would be performed.

As described above, the neural circuit tissue according to this embodiment can be used as a model of the brain that mimics the function of the brain. Therefore, for example, the neural circuit tissue may be brought into contact with a desired substance (NMDA inhibitor, AMPA inhibitor, psychotropic drug, etc.) to detect the effect on excitatory synaptic transmission or inhibitory synaptic transmission, or By detecting the effect on Phase-Amplitude Coupling (PAC), which is characteristic of various diseases, it is possible to screen for candidate substances that change the neural activity of the brain.
Furthermore, it is possible to screen for substances that may change short-term memory or long-term memory (such as K252a and anisomycin), especially substances that may enhance short-term memory. For example, in the presence of a certain substance, the duration of neural activity (duration of neural activity after light irradiation) in response to the light stimulation of the axon bundle of the neural circuit tissue according to the present embodiment is in the absence of the substance. If it is longer than in the case of, it can be determined that the substance may function effectively for the sustainability of short-term memory.

If this specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include more than one.
Hereinafter, the present invention will be described with reference to examples, but the present examples are merely examples of embodiments of the present invention, and do not limit the scope of the present invention.

1. 1. Materials and methods 1-1. Preparation of PDMS-MEA Chips SU-8 Master Positive Pattern Models were created using standard photolithography techniques (Bowen et al., Frontiers in Systems Neuroscience 13, doi: 10.3389 / fnsys.2019.00045. ECollection 2019).
SU-8 (2100 or 2075) was poured onto a silicon wafer (4 inches) and centrifuged (1200-1500 rpm, for 30 sec) to coat the surface. Wafers were pretreated on a hot plate at 65 ° C. for 90 minutes and heat treated at 95 ° C. for 40 minutes. After that, UV (365 nm, 2.5-3.0 mWcm ² ) was irradiated for 60-75 seconds using a photomask. The wafer was heat-treated on a hot plate at 65 ° C. for 7 minutes and then heat-treated at 95 ° C. for 13 minutes. After cooling the wafer, SU-8 was developed with SU-8 developer for 15 minutes and washed 3 times with isopropyl alcohol. The wafer was heat treated in an oven at 150 ° C. for 3 minutes. The thickness of SU-8 was about 150 μm.
Microfluidic devices were made using a polydimethylsiloxane (PDMS) silicone elastomer kit Sylgard184 (Dow Corning). Silicone elastomers and hardeners were mixed in a 10: 1 weight ratio, degassed, poured into patterned SU-8 structures and cured in an oven at 80 ° C. for 6 hours. Holes for organoids and reference electrodes were made with biopsy punches (1.5 mm and 2 mm, respectively). The glass ring used to store the medium (inner diameter: 22 mm, outer diameter: 25 mm) was glued to the PMDS device. The prepared PMDS device was sterilized in an autoclave and subjected to 70% ethanol treatment and UV treatment.

1-2. Human iPS cells Human iPS cells were obtained from Riken Cell Bank (409B2, HPS0076) (Okita et al., Nat Methods 8 409-412 2011). Cells were maintained on ESC-qualified Matrigel-coated 6-well plates on the first day in mTeSR plus medium (STEMCELL Technologies) supplemented with 10 μM Y-23632 (Wako for the first 24 hours only). Then, using ReLeSR (STEMCELL Technologies), the plants were planted every 5-7 days and then cultured.

1-3. Preparation of cerebral organoids In order to produce cerebral organoids, first, iPS cells were separated using TrypLE Express so as to become a single cell. 20,000 cells were then plated on a round-bottomed low-adhesion surface 96-well plate (Prime surface, Sumitomo bakelite) supplemented with mTeSR medium containing 10 μM Y-23632. After 24 hours, the medium was changed to nerve induction medium (DMEM-F12, 15% (v / v) knockout serum replacement, 1% (v / v) MEM-NEAA, 1% (v / v) Glutamax, 100 nM LDN- It was replaced with 193189 and 10 mM SB431542), and then the medium was replaced every two days. On the 10th day after culturing, the medium was 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement (without vitamin A), 1% (v / v) Glutamax, 0.5% (v / v). ) Replace with a 1: 1 mixture of DMEM / F12 and Neurobasal medium supplemented with MEM-NEAA, 0.25 mg / ml (v / v) human insulin solution and 1% (v / v) Penicillin / Streptomycin, followed by Cultured until the 18th day while exchanging the medium every 2 days, and the medium was maintained on the 18th day after the culture (0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement (vitamin A). Free), 1% (v / v) Glutamax, 0.5% (v / v) MEM-NEAA, 0.25 mg / ml (v / v) human insulin solution, 20 ng / ml BDNF, 200 mM ascorbic acid and 1% (v / v) Neurobasal medium supplemented with Penicillin / Streptomycin) was replaced. Brain organoids were then cultured for 4 weeks and used to make connected organoids.

1-4. Formation of Connected Organoids in PDMS-MEA Two brain organoids were cultured in PDMS-MEA and connected by axon bundles. Culturing for connecting brain organoids with axon bundles was performed by a method obtained by modifying the method disclosed in the previous report (Non-Patent Document 7).
The electrodes of the MEA (multi-electrode array) were aligned with the PDMS microchannel (cerebral organoid culture hole) and both were connected. Microchannels were coated in DMEM / F12 (1:30) with ESC-qualified Matrigel (Corning) for 1 hour at room temperature. The solution used for coating was replaced with maintenance medium. The brain organoid was then placed in a microchannel and gravitationally submerged to the bottom. The maintenance medium was changed every 2 days.

1-5. Multi Microelectrode Array (MEA)
Twenty-four hours before measuring neuronal activity with multiple electrodes, maintenance medium was added to 1% (v / v) B27 supplement (containing vitamin A), 1% (v / v) Glutamax, 20 ng / ml BDNF and 1%. (v / v) Replaced with Brainphys containing Penicillin / Streptomycin. PDMS-MEA was set in the MED64 system (Alpha MED Scientific) and electrical signals from all 64 electrodes were recorded at 37 ° C. for 5-30 minutes at a sampling rate of 20,000 Hz. Noise during recording of electrical signals was removed with a bandpass filter between 0.1-10,000 Hz. The unprocessed (raw) signal is further filtered through a bandpass filter (300-3,000 Hz) for spike analysis, raster plotting, spike clustering, or a low frequency path (<1000) for analysis of local field potentials. Hz). All post-hoc analysis was then performed using MATLAB Signal Processing Toolbox, Curve fitting Toolbox, Deep learning Toolbox, Parallel Computing Toolbox, and Wavelet Toolbox. All analyzes and calculations were performed using MATLAB software. All scripts for the calculations in this example were downloaded from https://github.com/TatsuyaOsaki/Matlab_function.

　式（１）中、aおよびbは、各々、スケール因子（1/Hz）およびマザーウェーブレット因子の中心位置を表す。また、式（１）中、G（x）はcomplex Morlet functionを表す：

　式（２）中、周波数バンド幅F_Bは5に設定し、中心周波数F_Cは1に設定した。 1-6. Wavelet coherence and wavelet transform for frequency separation Wavelet coherence is a measure of the correlation between two signals at a particular frequency. Wavelet coherence from LFP records was calculated by Eq. (1). f (t) was calculated using the functions cwt () and icwt () in the "Wavelet Toolbox":

In formula (1), a and b represent the center positions of the scale factor (1 / Hz) and the mother wavelet factor, respectively. Further, in equation (1), G (x) represents a complex Morlet function:

In equation (2), the frequency bandwidth F _B was set to 5 and the center frequency F _C was set to 1.

　上記式中、mはタイムラグ、Eは期待値演算子である。2つのシグナル間にライムラグが無い場合、以下の式で示すように、相関を正規化するためのscaleoptオプションを1とした。

1-7. Cross-correlation
The cross-correlation R was calculated by the xcorr () function in MATLAB. The two signal series, xn and yn Cross-correlation sequence, were calculated as shown by the following equation:

In the above equation, m is the time lag and E is the expected value operator. When there is no lime lag between the two signals, the scaleopt option for normalizing the correlation was set to 1 as shown in the following equation.

　P(S)はサイズSの雪崩を観察する確率である。αは指数で、log-log座標における相関の傾きを表し、κは比例係数である。 1-8. Neuronal avalanche A neuronal avalanche is an event characterized by a continuous pattern of neuronal activity within nerve tissue. The time bin (Δt) calculation was set to 3 milliseconds. The probability was calculated by the following formula:

P (S) is the probability of observing a size S avalanche. α is an exponent, represents the slope of the correlation in log-log coordinates, and κ is a proportional coefficient.

1-9. Optogenic Control of Connected Organoids An optogenetic tool was used to control the activity of connected organoids (see Figure 10 below). AAV-CAG-hChR2H134R-tdTomato was donated by Karel Svoboda (Addgene plasmid # 28017). pAAV-CAG- ArchT-GFP was donated by Edward Boyden (Addgene plasmid # 29777).
After mixing 5 μL of AAV viral vector with 500 μL of maintenance medium, the medium was replaced with medium in a PDMS-MEA chip 72 hours before MEA measurement. 470 nm Fiber-coupled LED for hChR ^2H134R (M470F3-470 nm, 17.2 mW (Min) Fiber-Coupled LED, 1000 mA, Thorlabs) and 565 nm Fiber-coupled LED for Arch-T (M565F3, 565 nm, 9.9 mW (Min) Fiber-Coupled LED, 700 mA, Thorlabs) was controlled by a T-cube high power LED driver (LEDD1B, 1.2A, Thorlabs). Light was irradiated with multimode fiber (0.22 NA, High-OH, φ105 μm Core, 250-1200 nm, Thorlabs). The TTL pulse was generated by Arduino and controlled by the LED driver. I downloaded the source code from https://github.com/TatsuyaOsaki/Arduino_optogenetics.

1-10. ScRNA Sequencing and Data Processing Single, fused, and connected organoids cultured on a PDMS-MEA chip for 7 weeks were extracted from the PDMS device and centrifuged at 100 xg for 30 seconds. To obtain a single cell suspension, the organoids were dissociated with AccuMax at 37 ° C. for 10-30 minutes and then centrifuged at 200 xg for 5 minutes. The pelleted cells were then resuspended in DMEM containing 10% FBS and subjected to 10x Genomics Chromium single-cell RNA-seq library preparation according to the manufacturer's protocol. Finally, the library was sequenced on DNBSEQ with 150 bp paired end reads. Sequencing data was processed with default parameters using the Cell Ranger analysis pipeline v3. Reads were aligned to the human reference genome (GRCh38). Cell Ranger output "filtered gene-bar coded" I loaded the count matrix into Scanpy (Wolf et al, 2018) and another python package (scanpy = = 1.81 anndata == 0.7.6 umap = = 0.5.1 numpy) == 1.19.5 scipy == 1.4.1 pandas == 1.1.5 scikit-learn == 0.22.2.post1 statsmodels == 10.10.2 python-igraph == 0.9.6 pynndescent == 0.5.4) Downstream Loaded for analysis of. Poor quality cells were excluded based on the criteria of min_genes> 200, min_cells <3, mitochondrial gene percentage <10%, nFeatures <4000. In addition, cells with a hemoglobin lead ratio of 5% or more were excluded. As a result, a total of 17,636 cells were finally analyzed. Principal component analysis (PCA) was performed using Scanpy to reduce the dimension of the data. UMAP was used to visualize the clustered data.

1-11. Identification of Neurons Participating in Axon Bundle Formation by the Fluorescent Protein Kaede In order to identify axon bundle-related neurons of connected organoids, the photoconverted fluorescent substance Kaede was gene-introduced into ligated organoids. First, AAV-CAG-Kaede by AAV-CAG-EGFP (Addgene plasmid # 28014) (Mao et al., Neuron 72, 111-123. 10.1016 / j.neuron.2011.07.029. 2011) and Coral Hue Kaede (pKaede-S1). A plasmid was constructed (see Figure 4A below). AAV-CAG-GFP was provided by Karel Svoboda (Addgene plasmid # 28014). AAV was made with AAVpro 293T (Takara). AAV was recovered using the AAVpro Purification kit midi (Takara) according to the manufacturer's protocol. AAV was infected with the connecting organoid at 4 to 6 weeks, and a photoconversion experiment was performed in the culture at 7 weeks. To visualize axon-related neurons of connected organoids, a 405 nm laser was applied to the axon bundle area and a Nikon confocal microscope (1.5 mm x 0.5 mm x 0.2 mm, 5x, total time: 60 min). , Nikon A1R). Then, the organoids were dissociated by Accumax at 37 ° C. for 10-30 minutes and centrifuged at 200 xg for 5 minutes. For flow cytometry, single cells were resuspended in PBS containing 1% BSA. Using BD FACS melody, Kaede red positive / Kaede green negative population and Kaede red negative / Kaede green positive population were collected as axon-related neurons and non-axon-related neurons, respectively. After sorting, total RNA was collected for RT-PCR analysis.

1-12. Human iPS cells were transfected with GFP fluorescent protein or mCherry fluorescent protein to visualize knock-in axonal elongation by electroporation with the CRISPR-Cas9 method of GFP fluorescent protein and mCherry fluorescent protein. The GFP fluorescent protein and mCherry fluorescent protein were inserted into the safe region site of AAVS1 (Adeno-associated virus integration site 1), respectively. The iPS cells were collected by TrypL Eexpress treatment and centrifuged to collect the cells. Then 5 μg of PX458-AAVS1 plasmid and 5 μg of AAVS1-Pur-CAG-EGFP plasmid (Varley et al., PLOS ONE 15, e0223812 2020) or 5 μg of AAVS1-Pur-CAG-mCherry plasmid (Varley et al., PLOS ONE 15, e0223812 2020) was mixed with 1 × 10 ⁶ cells in 100 μL Opti-MEM. Transfer the plasmid-cell mixture to NEPA cuvette (EC-002S) with a pipette and electroporate (Poring pulse (voltage: 125V, pulse length: 5 msec, pulse: 50 msec; a number of pulses: 2, decay rate 10%). ), Transfer pulse (voltage: 20V, pulse length: 50 msec, pulse: 50 msec; a number of pulses: 2, decay rate 40%)) was generated by NEPA21 electroporator (NEPA gene). Electroporated iPS cells were seeded in 4 wells supplemented with Matrigel-coated 6-well plates containing mTeSR plus (containing 10 μM Y-23632). After 24 hours, 0.75 μg / ml puromycin was added to the transfected cells and treated for 2 hours for selection. The cells were then subcultured and grown in two Matrigel-coated wells on a 6-well plate. PX458-AAVS1, AAVS1-Pur-CAG-EGFP and AAVS1-Pur-CAG-mCherry plasmids were provided by Dr. Adam Karpf and Dr. Su-Chun Zhang (Addgene 113194, 80945 and 80946).

1-13. Frozen sections and immunohistochemistry Cerebral organoids were fixed in 4% paraformaldehyde (PFA) and 8% sucrose at 4 ° C for 15 minutes and washed 3 times with PBS (each wash was incubated at room temperature for 10 minutes). ), Transferred to a 30% sucrose solution, and incubated overnight at 4 ° C. The sucrose solution was then removed and the brain organoids were equilibrated with OCT compound for 15 minutes at room temperature. Brain organoids were then embedded in the OCT compound on dry ice. Brain organoids were then stored at -80 ° C or 20 μm thick frozen sections were made.

Cells were fixed with 4% paraformaldehyde for 20 minutes and treated with 0.2% Triton X-100 for 5 minutes for permeabilization of cell membranes. Blocked with 1% bovine serum albumin (BSA) for 2 hours. The cells were then treated with the primary antibody for 2 hours at room temperature. In addition, it was treated with a secondary antibody for 2 hours at room temperature. As primary antibodies, mouse anti-neuron-specific βIII tubulin (Biolegend 801202, 1: 1200), rabbit anti-neuron-specific βIII tubulin (Sigma, ZooMAb, 1: 200), mouse anti-human PAX6 (DHSB, 1: 1200) 100), rabbit anti-human GAD67 (Santa Cruz, 1: 100), rabbit anti-human vGluT1 (Sigma, ZooMAb, 1: 200), mouse anti-human CTiP2 (Abcam, 1: 100) rabbit anti-human SATB2 (Abcam, 1: 100) Abcam ab51502, 1: 100) or rabbit anti-human MAP2 (Sigma, ZBR2290, 1: 200) was used. In addition, as secondary antibodies, Alexa Fluor 555 anti-rabbit IgG (H + L, Alexa Fluor 405 anti-rabbit IgG (H + L), Alexa Fluor 488 goat anti-mouse IgG (H + L), Alexa Fluor 488 goat anti-rabbit IgG (H + L) and hAlexa Fluor 647 goat anti-rat IgG (H + L) were used.
The nuclei were stained with Hoechst dye for 20 minutes at room temperature and rinsed 3 times with Dulbecco's Phosphate-Buffered Saline (D-PBS ⁺⁺ ) containing Ca ²⁺ and Mg ²⁺ .
All cells and samples were observed under a fluorescence microscope (Axio Observer, Zeiss) or a confocal laser scanning microscope (Zeiss).

1-14. RT-PCR (Real-time reverse-transcription)
Total RNA was isolated from tissues using TriPure (Sigma) to measure the biological activity of brain organoids. Reverse transcription was performed using KOD One (Toyobo). The primer sequences are shown in Table 1. RT-PCR was performed at CFX Connec using KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems). In all experiments, the mRNA expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. RT-PCR was performed at least 3 times using cDNAs prepared from different tissues.

1-15. Calcium (Ca ²⁺ ) Imaging A Ca ²⁺ indicator (GCaMP6f controlled by the CAG promoter) was transfected with AAV1 to visualize neuronal activity in fluorescence. pAAV.CAG.GCaMP6f.WPRE.SV4 was donated by Douglas Kim & GENIE Project (Addgene # 100836).
After mixing 5 μL of AAV viral vector with 500 μL of maintenance medium, the maintenance medium was replaced with medium in PDMS-MEA 3 days prior to measurement. After treating the AAV mixture for 6-12 hours, the medium was replaced with fresh maintenance medium. 30 minutes before measurement, maintenance medium 1% (v / v) B27 supplement (containing vitamin A), 1% (v / v) Glutamax, 20 ng / ml BDNF and 1% (v / v) Penicillin / Streptomycin Was replaced with Brainphys. Next, the device was set on the stage of the microscope (the configuration of the microscope is shown in FIG. 12 described later). Time-lapse images were saved at frame speeds of 20 fps and above in 10 minutes. Data analysis was performed using MATLAB (MathWorks). Regions of interest (ROIs) manually surrounded the cell bodies of neurons in organoids. The baseline fluorescence value of each ROI was the average of the lowest baseline values of the sample. ΔF / F was calculated as (F − F0) / F0 × 100. Where F is the instantaneous fluorescence intensity from the unprocessed ROI time series image.

2. Results First, the results of this embodiment shown in the drawings will be described.

FIG. 1 shows the results of evaluating the formation and characteristics of connecting organoids on a PDMS-MEA chip. A is a schematic diagram of the connecting organoids on the PDMS-MEA chip. The two brain organoids were cultured in two chambers cross-linked by microchannels on the chip. B shows an example of a PDMS-MEA chip. The PDMS-MEA chip is composed of a MEA probe (MEA), a PDMS (PDMS microfldic layer), a glass solution storage ring (Reserviro ring), and a PDMS lid (Lid) (i and ii). Under each cerebral organoid, electrodes (4 × 4 array) composed of 16 metal thin films and the like are installed (iii). C shows a typical internal structure after 4 weeks (upper figure) and 8 weeks (lower figure). Scale bar: 150 μm. D shows the gene expression profile of brain organoids cultured for 2 to 10 weeks. The vertical axis shows the gene name, and the horizontal axis shows the time (week) after culturing. In E, the axon extended from one organoid to another organoid at 5 weeks, and the axon bundle was formed by 6 weeks. From the top, fluorescent images of brain organoids 4 weeks, 5 weeks (axon elongation) and 6 weeks (organoid connection by axon bundles) after culturing iPS cells are shown. F indicates the change over time in the thickness of the axon bundle. The vertical axis is the thickness of the axon bundle, and the horizontal axis is the culture time (weeks) from iPS cells (n = 6). G indicates the abundance ratio of excitatory neurons (VGLUT1 antibody staining positive) and inhibitory neurons (GAD67 antibody staining positive) in organoids. The vertical axis is the cell density, and the horizontal axis is the culture time (weeks) from iPS cells (n = 3). H indicates the result of immunohistological analysis of organoids. After culturing iPS cells for 8 weeks, immunohistological analysis revealed the presence of layered structures within the connecting organoids. Immunostaining for PAX6 and CTIP2 shows proliferative and cortical sublayers, respectively. I is a schematic representation of a method of recording neuronal activity from connecting organoids on a PDMS-MEA chip. The raw analog signal from the electrodes was amplified at a sampling rate of 20 kHz and converted to a digital signal (16 bits). After that, the signals are 300-3,000Hz bandpass filter (Bandpass filter) for spike analysis and 1,000Hz low frequency bandpass filter (Lowpass filter) for local field potential (LFP). Processing was performed. J shows a representative example of a connecting organoid 5 weeks after culturing iPS cells. The scale bar is 1 mm (i). In addition, examples of filtered signals from four typical electrodes under each organoid are shown. LE is the signal of the left brain organoid, and RE is the signal of the right brain organoid (ii). Wavelet coherence between the signals derived from the left and right organoids is shown (iii). K represents a representative example of the connecting connectoid 5.5 weeks after iPS cell culture. The scale bar is 1 mm (i). Burst-like activity synchronized with dense spikes and left and right organoids was detected from multiple electrodes (ii). Wavelet coherence showed a strong association between the two connecting connectoids (iii). L indicates the result of analyzing the synchronization of activities between organoids. The synchronization of neuronal activity of the two organoids increased during the culture period. The vertical axis shows the synchronicity index, and the horizontal axis shows the iPS cell culture time (week). M indicates the result of measuring the frequency of burst activity. The frequency of burst-like activity increased significantly depending on the culture period. The vertical axis shows the burst frequency, and the horizontal axis shows the iPS cell culture time (week). N shows the neuronal activity signals of the two connecting organoids (middle figure) and their enlarged views (lower figure). The figure above shows a raster plot. A time lag (Burst delay) was observed in the burst-like activity synchronized with the left and right organoids. O indicates the time lag between the bursts of the left and right organoids at different culture points. The vertical axis shows the burst delay, and the horizontal axis shows the iPS cell culture time (week). In addition, n = 20. * P <0.05, ** p <0.01; one-way ANOVA, error bar indicates SD (standard deviation).

FIG. 2 shows the results of analysis of neuronal activity of brain organoids connected by axon bundles. A shows the local field potential (LFP) signals in the 0.2-0.5 Hz band, 0.5-4 Hz (δ) band, and 30-300 Hz (γ) band extracted by the inverse continuous wavelet transformation. .. Eight weeks after culturing iPS cells, the connecting organoid generated low frequency oscillations in the 0.5-4 Hz (δ) band. B indicates the power integration result for each frequency band. The vertical axis shows the integrated value of wave power, and the horizontal axis shows the time (week) after culturing. C represents a representative example of the signal of neuronal activity of connecting organoids, single organoids and fused organoids. D indicates the burst frequency of various organoids. Single organoids, Fused organoids and connected organoids show the results of single organoids, fused organoids and connected organoids, respectively (n = 10). E shows the result of the inverse continuous wavelet transform in the 0.2-0.5 Hz band, the 0.5-4 Hz (δ) band, and the 30-300 Hz (γ) band. Oscillations in the δ band were detected in connecting organoids but not in single and fused organoids. In the figure on the right, for each wavelength band, the left bar graph shows the results of single organoids, the middle bar graph shows the results of fused organoids, and the right bar graph shows the results of connected organoids. F indicates the coefficient of variation of the burst frequency of the three types of organoids. The vertical axis shows the coefficient of variation of the burst interval, and the horizontal axis shows the post-culture time (week). G indicates the relationship between the volume of organoids (horizontal axis) and the average burst frequency (vertical axis).

FIG. 3 shows the results of genetic analysis of linked brain organoids (connecting organoids) using Single cell RNA seq. A shows 17,636 UMAP plots. Leiden clustering was performed on single, fused, connected organoids. (I) The cell population was classified into 14 clusters. (Ii) UMAP plots and density plots are shown for each sample. B and D show heatmaps of the top 30 genes that are statistically significant in each cluster. By known markers, the 14 clusters were further divided into four groups (Group 1 "NPC", Group 2 "Intermediate", Group 3 "Neurons", Group 4 "Other"). C is the result of normalizing the cell proportions of clusters 8, 6, 12, and 4 of single, fused, and connected organoids. It was found that the number of cells in cluster 8 of connected organoids was significantly higher than that of single and fused organoids, and the number of cells in clusters 6 and 12 (NPC population) of connected organoids was slightly lower than that of single organoids. E is an annotated UMAP plot with a known marker gene. F is a classification result by cell type by a known marker gene. G shows a UMAP plot of vGlut1 for visualization of excitatory neurons and DLX6 and GAL for visualization of inhibitory neurons. H indicates a UMAP plot of GRIA1 and GRIA2 that are highly expressed in cluster 8. I and J show plots of GRIA2 expression in cluster 8 of single organoids, fused organoids, and connected organoids. The average expression of GRIA2 in the linked brain organoids was higher than that of the single and fused organoids. Among the cluster 8, GRIA2 was highly expressed in DCX-positive cells (J).

FIG. 4 shows the results of characterizing axon bundles using a photoconverted fluorescent protein. A shows a plasmid map of a pAAV backbone plasmid expressing Kaede whose fluorescence wavelength is variable by UV irradiation by a CAG promoter. Kaede green fluorescent protein can be converted to Kaede red fluorescent protein by ultraviolet irradiation. One week after introducing the cerebral organoid into the microfluidic device, it was infected with AAV-CAG-Kaede. On the 49th day (7th week of culture), the axon bundle was irradiated with ultraviolet rays (405 nm laser, equipped with a confocal microscope). The cells were then isolated with a cell sorter to identify neurons involved in axon bundle formation (Kaede red positive) and neurons not involved in axon bundle formation (Kaede green positive) (see B). .. C shows a confocal microscope image of the articulated organoid before and after irradiation with ultraviolet rays. Kaede green is rapidly converted to Kaede red by UV irradiation. After that, Kaede red rapidly diffused and distributed in the axon bundle in the anteroposterior direction, and a gradation of Kaede green and Kaede red was formed in the axon bundle. D shows a cross-sectional view of the axon bundle and XY cross-sectional views below and above. In the central part of the axon bundle, most of the Kaede green was converted to Kaede red. It was also found that Kaede red in the axon bundle spreads not only to the lower part of the linked brain organoid but also to the perikaryon located at the upper part. E shows the distribution of Kaede red in axon-related neurons by three-dimensional reconstruction. F shows the result of quantifying the fluorescence intensities of Kaede green and Kaede red corresponding to the distance from the center of the axon bundle before and after ultraviolet irradiation. Kaede red was concentrated on the closest side of the axon bundle (see G). H indicates the distribution of Kaede green and Kaede red in the z direction of the linked organoids. It was observed that Kaede red (neurons extending axon bundles) was not localized at the bottom of the glass plate (see I). In addition, nerve cells with axon extension and nerve cells without axon extension were separated and quantified by flow cytometry. The average proportion of axon-related neurons was 32% and that of non-axon-related neurons was 68%, correlating with confocal microscopy images (see J). K indicates a change in gene expression relative to axon-stretching neurons relative to non-axon-stretching neurons. Genes such as CFOS, TBR1, vGLUT1, and NEAT1 were highly expressed in nerves derived from axon bundles, but DLX5, GAD1, and BCL11B were expressed. From the results of the scRNA-seq scatter plot (L) and UMAP plot (M), it can be inferred that NEAT1 and GRIA2 co-expressing cells are predominant in cluster 8 and that they are neurons involved in axon bundle formation. N shows a summary of the results obtained from the Kaede experiment and scRNA-seq. * P <0.05, ** p <0.01; student's t-test, error bar indicates SD (standard deviation).

FIG. 5 shows the results of a study on optogenetic inhibition of burst activity between connected organoids and synchronization between both organoids. A shows the configuration of an optogenetic device for inhibiting synaptic interactions between left and right organoids via axons. In addition, i shows a schematic diagram of a microfluidic device for optogenetic control, and ii shows a state in which ArchT is expressed in a connecting organoid by AAV and irradiated with light. The fiber optics can be moved and placed so that the organoids and / or axon bundles can be selectively illuminated via a fiber guide in which the optical fibers are installed, but here the distance between the axon bundles is 100 μm. It was placed and placed vertically. MEA was measured by connecting a 470 nm or 565 nm LED and a pulse generator (Arduino) to the optical fiber. The timing of light irradiation and the signal from a typical channel from the MEA amplifier were recorded by TTL logger, and the TTL signal and neural activity were synchronized at the time of analysis before analysis (Analysis Department) (see iii). ). The curved structure as a PDMS lens helped to focus the light on the axon bundle (see B). C shows the LFP and raster plots detected from the left and right organoids of the connecting organoids with or without light irradiation. The synchronized burst frequency was approximately 0.65 Hz when unirradiated with light, but decreased to almost 0 when irradiated with light (see D, n = 8). E indicates wavelet coherence between the left and right organoids of the connecting organoid. Light irradiation eliminated the low frequency oscillations, i.e., the associated activity between the organoids (n = 8). In addition, the synchrony between the regions measured by the δ-phase-δ-phase coupling was reduced by light irradiation (see F, n = 8). Light irradiation completely suppressed these synchronized burst activities (G, n = 5 from 3 independent samples). H is the result of calculating the total number of single spikes for 5 minutes. Light irradiation induced an increase in the number of spikes. Optogenetic inhibition of interregional interactions resulted in persistence of the brain hypothesis (see I). Note that * p <0.05, ** p <0.01; one-way ANOVA, and error bars indicate SD (standard deviation).

FIG. 6 shows the results of analysis of complex activities occurring in connected organoids using Phase-Amplitude Coupling and a hidden Markov model. A shows the raw (untreated) LFP plots detected from each connecting organoid at 9 weeks of culture of iPS cells. B indicates wavelet coherence between the two organoids. C indicates the modulation index of the phase amplitude coupling in the δ-phase / γ power and the θ-phase / γ power of the connected organoids at the 5th, 7th, and 9th weeks of culture. D is the delta-phase / γ-power and θ-phase / γ-power PAC modulation index of single organoids (“S” in the figure), fused organoids (“F” in the figure) and connected organoids (“C” in the figure). modulation index) is shown. E indicates the PAC modulation index within or between organoids in the connecting organoid. F outlines the brain hypothesis analysis of the left and right organoids. In addition, the extraction results of the brain hypothesis cascade are shown. The brain hypothesis was calculated based on the signals from 8 electrodes. The single spike cascade was analyzed on a 3 msec scale size. G shows a logarithmic plot of brain avalanche size and probability of occurrence at 5, 5.5 and 8.5 weeks after culture. H indicates the number of hidden patterns in the brain hypothesis at 5, 5.5 and 8.5 weeks after culture. I, J, K and L are the neural activities of connecting organoids treated with various neuromodulatory compounds (CNQX, APV, Bicculline, Baclofen, Buprenorphine, Clozapine and Diazepam). The result of comparing the patterns is shown. The average number of spikes (I), burst frequency (J), integrated power in the δ band (K), and PAC modulation index (L) were calculated. note that. * p <0.05, ** p <0.01; one-way ANOVA, error bar indicates SD (standard deviation).

FIG. 7 shows the analysis results of the short-term memory mechanism in the connecting organoid. A shows a schematic diagram of an optogenic stimulation experiment. Optogenic stimulation of axon bundles at 0.5, 1 and 1.5 Hz with a 470 nm laser source or LED induced synchronized burst activity. The effect of the stimulus persisted even after discontinuation of light irradiation. B and C show the measurement result of the burst frequency modulated by the light stimulus. There was a time lag between the photostimulation and the modulation of the burst frequency. D indicates a logarithmic plot (vertical axis) of the brain hypothesis size (horizontal axis) and appearance probability before stimulation (Before Stim.), During stimulation (During Stim.), And after stimulation (After Stim.). E indicates a time course of burst frequency stimulated at 1 Hz for 20 minutes (i) and 5 minutes (ii). The vertical axis shows the burst frequency, and the horizontal axis shows the time (minutes) from the start of stimulation. F indicates the result of measuring the duration of burst activity after light stimulation (20 minutes or 5 minutes). The duration is the time until the burst frequency after discontinuation of light stimulation decreases to 75% of the maximum burst frequency. G indicates the time lag (Delay time) from the light stimulation to the burst induction activity. When the connecting organoid was stimulated for 20 minutes, the time lag from the start of the light stimulation to the induction of the burst was significantly reduced compared to the first stimulation, and the time lag after the second and third stimulations was significantly reduced. H indicates the time course of burst frequency in the presence of K252a or anisomycin. I stimulates the duration of burst activity of connecting organoids under conditions without compound treatment (control) or with compound (K252a or Anisomycin) treatment (1st (1st), 2nd (2nd) and Shown every 3rd (3rd)). J indicates the time lag from the start of photostimulation to the induction of burst in the presence of K252a and anisomycin for each stimulus. FIG. K is a diagram showing the relationship between the number of hidden patterns of a nerve avalanche and the size of hidden patterns of a nerve avalanche. L indicates the result of examining the probability of occurrence of a neurological avalanche. Results for each stimulus (1st (1st), 2nd (2nd) and 3rd (3rd)) in the presence of control, K252a, and anisomycin (on: with photostimulation, off: without photostimulation) .. M indicates the result of examining the total number of hidden patterns of the brain hypothesis. Results for each stimulus (1st (1st), 2nd (2nd) and 3rd (3rd)) in control, in the presence of K252a and in the presence of anisomycin (Before: before photostimulation, on: during photostimulation (Before: before photostimulation, on: during photostimulation ( Left bar graph of each stimulus), off: After photostimulation (right bar graph of each stimulus)). N and O represent the fractal dimension of the LFP signal. Results for each stimulus (1st (1st), 2nd (2nd) and 3rd (3rd)) in control, in the presence of K252a and in the presence of anisomycin (Before: before photostimulation, on: during photostimulation (Before: before photostimulation, on: during photostimulation ( Left bar graph of each stimulus), off: After photostimulation (right bar graph of each stimulus)). Note that * p <0.05, ** p <0.01; one-way ANOVA, and error bars indicate SD (standard deviation).

FIG. 8 shows the results of analyzing various burst activity patterns supported by CaMKII-dependent signals. FIG. A is a diagram showing an example of burst activity induced by optogenic stimulation and neuron-derived potential distributions in the right-sided organoid and the left-sided organoid. 891 Burst traces are shown. The light-stimulated spikes and burst waves continued after the photostimulation. Secondary and tertiary waves of burst activity were also observed. B indicates the time lag (ms) after the light stimulation (1st, 2nd and 3rd) until the burst activity is induced. Measured in the presence of control, K252a or anisomycin. In the presence of controls and anisomycin, repeated photostimulation (2nd and 3rd) significantly reduced lime lag from inducing to burst activity. In contrast, no reduction in lime lag was observed in the presence of K252a (n = 3). (I) of C shows a histogram in which the induced burst wave and the kernel density estimation (line graph) are superimposed in the presence of control, K252a or anisomycin. Repeated light stimulation increased the complexity of burst activity. (Ii) of C is a figure which shows the ratio of the induced burst activity with a 2nd peak and the induced burst activity without a 2nd peak. Results are shown in the presence of Control, K252a or anisomycin. D indicates a violin plot at the peak of burst activity (n = 5). E represents a representative example of crosstalk between each organoid of the connecting organoids in self-induced burst activity. F is the result of quantifying diversity by calculating the entropy of burst activity PCA (n = 3). * P <0.05, ** p <0.01; one-way ANOVA or student's t-test, error bar indicates SD (standard deviation).

FIG. 9 is the result of visualizing and analyzing the elongated axons of the connecting organoids. A shows a schematic view of a method of knocking EGFP and mCherry under the control of a CAG promoter into the AAVS1 region of iPS cells (left figure) and a fluorescent image of a fluorescent protein expressed in iPSC cells (right figure). GFP-labeled or mCherry-labeled brain organoids were generated to visualize axon elongation on PDMS chips. B shows the result of comparing the maximum axon length of the wild-type brain organoid with the GFP-labeled or mCherry-labeled brain organoid (n = 3). The vertical axis of the graph on the right shows the length of the most elongated axon (μm), and the horizontal axis shows the culture time (days) of iPS cells. C is an image of observing the elongation of axon bundles between organoids placed at different intervals (2 mm, 3 mm, 4 mm, 5 mm) at 5 and 6 weeks after culturing iPS cells. show. D indicates the result of measuring the thickness of the axon bundle extending between the organoids placed at different intervals (2 mm, 3 mm, 4 mm, 5 mm). The thickness of the axon bundle was measured at the center of the microchannel. Axon bundle thickness of GFP-labeled connecting organoids (n = 3). E shows a 3D confocal microscope image of the GFP-labeled connecting organoid. Brain organoids labeled with GFP and mCherry were ligated in a microfluidic device. Axons labeled with GFP or mCherry extended to mCherry-labeled brain organoids and GFP-labeled organoids, respectively, on the chip after 2 weeks via overlapping thick axons (F). G indicates the result of counting the number of axons that have reached other brain organoids. The vertical axis shows the number of axons reached by the organoid, and the horizontal axis shows the iPS cell culture time (day). H is a graph showing the relationship between the thickness of the axon bundle and the frequency of synchronized burst activity. The frequency of synchronized burst activity increased as the thickness of the axonal bundle increased. I is an image showing the state of axon extension toward the other organoid. Immunostaining of SynI revealed synaptic connections between organoids. ** p <0.01; one-way ANOVA or student's t-test, error bar indicates SD (standard deviation).

FIG. 10 shows the results of comparing the neural activities of single organoids, fused organoids, and connected organoids. A is a schematic diagram of a procedure for producing a single organoid, a fused organoid, and a connected organoid. All brain organoids were made in the same way until day 21. To make fused organoids, two brain organoids were placed in one well of a poorly adhesive 96-well plate. Then, on day 28, single and fused organoids were placed on the MEA probe. Also, on day 28, organoids were placed on PDMS-MEA probes to generate connecting organoids. After culturing on the MEA probe for 2 weeks, neuronal activity was measured. B shows a representative example of a single organoid placed on the MEA probe (left figure). Periodic and synchronized neuronal activity was detected in a single organoid (right figure). C shows a representative example of the fusion organoid placed on the MEA probe (left figure). More active and synchronized neuronal activity was detected in fused organoids compared to single organoids. D shows an outline of a method for detecting a time difference in neuronal activity within an organoid (Intra-) and between organoids (Inter-) by an electrode. E shows the result of quantifying the time lag of neuronal activity detected from the electrode (n = 4). The inter-connected time lag was significantly smaller than the inter-fused time lag. F indicates the result of measuring the signal transduction rate. Inter-connected signal transduction was faster than signal transduction between the other two points (n = 4). * P <0.05, ** p <0.01; one-way ANOVA or student's t-test, error bar indicates SD (standard deviation).

FIG. 11 shows the results of measuring organoids connected by axon bundles of various lengths and their axon velocities. A shows an example of three types of microfluidic chips (5.5 mm, 7.8 mm and 12 mm) with different lengths between channels. Arrows indicate the location of organoids. Conduction lag times were plotted against distance to estimate axon conduction velocity (B). The delay coefficient was 65 msec. The axon conduction velocity was approximately 2 mm / sec (n = 4).

FIG. 12 shows the results of analysis of neuronal activity in organoids in which axon bundles are physically cleaved. After physically cleaving the axon bundle (A) between the connecting organoids (B), the neuronal activity detected in each organoid (left organoid and right organoid) was measured. C shows the result of measuring the wavelet coherence of the disconnected organoid. From this result, it was clarified that the truncated organoids had almost no synchronized activity.

FIG. 13 shows the results of drug treatment for connecting organoids. A indicates the LFP signal of the connecting organoid at different culture periods (5.5 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks and 8.5 weeks). B is the result of measuring the signal propagation rate in the presence of the drug. From this result, it was found that the drug did not affect the transmission rate (n = 4). C indicates the result of measuring the amplification of spikes in the presence of a drug (n = 4).

FIG. 14 shows a scalogram of the LFP signal and wavelet transform of the connecting organoid in the presence of various agents.

FIG. 15 shows the results of simultaneous measurements of Ca ²⁺ transients and electrical activity. (I) of A shows the configuration of an optical device for performing Ca ²⁺ imaging and MEA recording of connecting organoids. Three days prior to AAV2 measurements, the connecting organoid was transiently transfected with the Ca ²⁺ reporter gene (GCaMP6f) (see ii and iii). Then, at the same time as capturing the temporal image with a microscope device, the LFP activity from MEA was simultaneously acquired. B showed the firing pattern of neurons in the connecting organoid at 7 weeks after culturing iPS cells by a trace image of the calcium response. Synchronized burst activity was observed between the left and right organoids (see C). The burst time lag between the two connected organoids was consistent with MEA recordings. D shows a plot of Ca ²⁺ concentration changes and MEA signal recordings. The two signals corresponded to each other. Calcium imaging and MEA records were consistent during burst activity (see E). F and G are the results of plotting the correlation coefficients obtained from the 12 neurons of the organoid on the left side g and the 12 neurons of the organoid on the right side for each nerve. Ca ²⁺ transient activity was correlated between organoids as well as within each organoid. On the other hand, if the signal is intentionally shifted by 50 m sec (1 frame), the correlation between the organoids becomes stronger, indicating that the left and right organoids are active with a delay of about 50 m sec. rice field.

The results of this embodiment will be described in detail below with reference to the results shown in the above drawings.

2-1. Neuronal activity of interconnected brain organoids on the PDMS-MEA chip Many regions of the brain are connected by interconnected axons. To model the simplest macroscopic neural circuit, a pair of brain organoids derived from human iPS cells were cultured in microfluidic culture chips (FIGS. 1A and 1B). The PDMS-MEA chip consists of a multi-electrode array (MEA) layer, a polydimethylpolyolefin (PDMS) microfluidic layer, and a ring and lid of a culture tank. The PDMS layer is provided with a pair of holes for containing and culturing each brain organoid. These two holes are connected at opposite ends by a spatial flow path structure that guides organoid axons to each other. Four weeks after initiation of culture to differentiate human iPS cells into cerebral organoids, cerebral organoids express neural markers (eg, DCX and TUBB3) and cortical layer-related genes (eg, TBR1 and SATB2). From this (Fig. 1C and D), it was confirmed that the brain organoids were successfully differentiated. After 4 weeks of culture, brain organoids were placed on PDMS-MEA. The two brain organoids were connected by a thick axon bundle within 6 weeks (within 2 weeks of placement on the chip) (Fig. 1E). On the chip, GFP-expressing brain organoids and mCherry-expressing brain organoids "hand-shake" type connections were shown (FIGS. 9A-I). Two brain organoids were connected to other organoids with as many axons as each other (Fig. 9G). The thickness of the axon bundle was about 75 μm after 6 weeks and about 120 μm after 8 weeks (4 weeks after placement on the chip) (Fig. 1F). vGlut1 positive excitatory neurons and GAD67 positive inhibitory neurons were contained in about 70% and 5-10% of the cells in the brain organoid, respectively (Fig. 1G). In the brain organoids connected by axon bundles, a layered structure was observed under the cortex (Fig. 1H).
As described above, it is considered that the two brain organoids can be connected to each other by axons by culturing, and as a result, the functions of the brain generated during the developmental process can be imitated.

In addition, neuronal activity (spontaneous firing activity) was detected from the electrodes installed under each brain organoid (Fig. 1B and I). Action potential spikes and local field potentials (LFPs) were extracted by high frequency filters and low frequency filters, respectively. After 5 weeks (1 week after culturing on the chip), neuronal activity was detected in each brain organoid (Fig. 1J). At this point, the activities of the two brain organoids were not synchronized and the axonal connections between the brain organoids were not sufficient (Fig. 1E). After 5.5-6 weeks (1.5-2 weeks after culturing on the chip), synchronized burst-like activity was observed between the two brain organoids (Fig. 1K, arrow). The burst-like activity of each brain organoid increased rapidly, and the time when both burst-like activities were synchronized and the time when the two brain organoids were connected via the axon bundle were almost the same. It was observed that continued culture of cerebral organoids increased the frequency of neuronal activity synchronization and the frequency of burst-like activity (FIGS. 1L and M). The thickness of the axon bundle and the frequency of neural activity showed a positive correlation (Fig. 9H). This result suggests that axon bundles promote neural activity in connecting organoids. In addition, the signals from the two connected brain organoids were detected with a short time (about 100 ms or less) shifted (FIGS. 1N and O). This result suggests that spontaneous neural activity occurred in one brain organoid and propagated through the axon bundle to the other brain organoid with a slight time lag. Furthermore, the above results also show that the two brain organoids induce signal propagation with each other and the connections are functionally bidirectional.

2-2. Complex neuronal activity induced by axonal connections between brain organoids 6 weeks after iPS cells started culturing (2 weeks after culturing on chips), slowly undetected before axonal connections were established The LFP signal was detected in each cerebral organoid (Fig. 2A). It is known that clustered action potentials of multiple neurons produce coordinated low-frequency LFP patterns. After an additional week of culture on the chip (7 weeks after the start of iPS cell culture), the LFP pattern of connecting brain organoids became more complex, with strong action potentials appearing in the δ wavelength band (0.5-4 Hz) (5-4 Hz). 2A and 2B). Based on previous reports (Trujillo et al., Cell Stem Cell 25 558-569.e7 2019) showing that cerebral organoids produce action potentials in the δ wavelength band after several months of culture, the brain is relatively early. It was not expected that strong and complex action potentials would be detected in organoids. Therefore, we compared the neuronal activity of "connected (connected by axon bundles)" organoids and conventional brain organoids ("single organoids") in this study. Since connecting organoids contain almost twice as many cells as a single organoid, we decided to evaluate the neuronal activity of tissues that directly fused the two organoids (“fused” organoids) as an additional control. (Fig. 2C and Fig. 10A). After culturing iPS cells for 6 weeks, oscillatory neuronal activity was detected in all brain organoids (FIGS. 2C, 10B and C). More frequent burst-like activity was observed in fused organoids compared to single organoids, suggesting that the total number of neuronal cells in the organoid affects neural activity. The frequency of burst-like activity from connecting organoids was significantly higher than that of single or fused organoids, but the number of neurons contained in the fused organoid and the connecting organoid was about the same (Fig. 2D). ). These findings suggest that axonal connections between organoids enhance neuronal activity of cerebral organoids.

In order to understand the network structure of organoids, we analyzed the signal propagation rate calculated from the distance between two electrodes and the time lag of the signals detected from these electrodes. Signal transduction rates between fused organoids were slower than within a single organoid (FIGS. 10E and F), while signaling rates between connecting organoids were faster than within a single organoid (FIGS. 10E and F). F). The delay in signal transduction between organoids (time lag) was significantly greater in fused organoids than in connected organoids. The rate of transmission is so fast that changes in axon length do not affect the delay in signal transduction (below the measurement limit) (Fig. 11), and the results show that the composition of synaptic connections within the organoid signals. It was suggested that it determines the transmission rate. These results suggest that axonal connections between organoids serve as "highways" of signals between connecting organoids and promote neuronal activity.

The activity of connected organoids was concentrated in the δ wavelength band (0.5-4 Hz) compared to single organoids and fused organoids (Fig. 2E). To quantify the temporal complexity of neuronal activity in another way, we evaluated the periodicity of burst-like patterns of organoids. The coefficient of variation of the frequency of burst-like neuronal activity (CV, showing variability; standard deviation / arithmetic mean) was significantly higher for connected organoids when compared to single and fused organoids (Fig. 2F). ). This suggests that burst-like neuronal activity in connecting organoids is significantly more irregular and complex than burst-like neuronal activity in single and fused organoids. CVs of connecting organoids were consistently increased from iPS cell culture to 9 weeks, but CVs of single and fused organoids showed no significant variation during this culture. Burst activity of fused organoids was more frequent, consistent with their size, compared to activity of single organoids (Figure 2G), leading to increased numbers of neurons and synapses in tissues. It seems to be dependent. On the other hand, connecting organoids had a higher frequency of burst-like neuronal activity compared to single and fused organoids, regardless of their size. These results indicate that connecting organoids constitute more complex neural circuits than single or fused organoids, and that interconnection between organoids induces complex neuronal activity in cerebral organoids. ..

2-3. Analysis of gene expression in connected organoids by single cell RNA-seq We evaluated how interconnection between organoids alters the gene expression profile in organoids by single cell RNA seq. Single-cell (sc) RNA-seq of single organoids, fusion organoids, and connecting organoids at 7 weeks of culture was performed. 17,636 single cells aggregated from all three conditions were subjected to principal component analysis (PCA) and then visualized as UMAP plots (Fig. 3A (i)). The gene expression profiles of single organoids, fused organoids, and connected organoids were found to be generally similar with slight differences (Fig. 3A (ii)). The cells were isolated into 14 clusters divided into 4 groups ("NPC", "intermediate", "neurons", "other" clusters) (Fig. 3B, heatmap the top 30 statistically significant genes). (Plotted in). According to known markers (eg HES1, DCX, TBR1), "NPC" (clusters 6, 12, 13), "Intermediate" (clusters 1, 2, 3, 11), "Neurons" (clusters 4, 5, 7) , 8, 9, 10), and "Other" (cluster 0) were determined (Fig. 3C).

Density plots for single organoids, fused organoids, and connected organoids revealed that there was variation in cell population in each of the three samples (Fig. 3A (ii)). In particular, clusters 1, 4, and 8 were found to be more abundant in connecting organoids compared to single and fused organoids. In addition, in the connected organoids, the number of cells classified into NPC clusters 6 and 12 was smaller than that of single organoids and fused organoids (Fig. 3C). Furthermore, when annotated by cell classification by genes with different expression levels, it was found that clusters 4 and 8 may be involved in mature excitatory neurons expressing neurotransmitters (Fig. 3B). They were also classified into "excitatory neurons" (clusters 4 and 8) and "inhibitory neurons" (clusters 9 and 10), which are characterized by the expression of VGLUT1 and DLX6, respectively (FIGS. 3E and G). The ratio of excitatory neurons to inhibitory neurons in connecting organoids was about 85:15, which was almost the same as that of single organoids and fusion organoids (Fig. 3G). This result was almost in agreement with the result by immunostaining of cerebral organoids (Fig. 1H). Furthermore, GRIA2, which is a glutamate ion receptor AMPA type subunit II, is highly expressed in the neurons of cluster 8, but GRIA1 is predominantly expressed in other clusters (Fig. 3H), and that of cluster 8 Nerve cells have been shown to follow the maturation process that occurs in vivo (Balik et al., 2013). In addition, the expression level of GRIA2 in the connecting organoids of cluster 8 was higher than that of single organoids and fused organoids (Fig. 3I). This indicates that connecting organoids have more mature neurons than single or fused organoids, and these neurons are more mature with connecting organoids. In particular, among the nerve cells expressing DCX, the expression level of GRIA2 in the linked organoid was increased (Fig. 3J), and as previously suggested, the increased activity of the nerve cell resulted in the linked organoid. It was suggested that nerve cells may show more maturity in (Gordon et al., Nature Neuroscience 24 331-342 2021). Gene-Ontology analysis revealed that neurons in cluster 8 express various types of receptors and potential gate channel-related genes (GRIN2B, CANA1A, CANA1E, SCN2A, NTRK2).

2-4. Identification of axon-specific cell populations using Kaede In order to identify nerve cells that extend axons from cerebral organoids, we used the fluorescent protein Kaede whose fluorescence wavelength changes with light irradiation (Fig. 4A). Kaede (Kaede-green) can be immediately converted to red-fluorescent Kaede (Kaede-red) by UV irradiation (Ando et al., Proc. Acad. Natl. Sci., 99 12651-12656 2002). At 5 weeks of culture, the connected organoids were infected with Kaede-expressing virus (AAV-CAG-Kaede). Then, at the 7th week of culturing, ultraviolet irradiation was performed with a confocal microscope to perform light conversion (FIG. 4B). Prior to UV irradiation, Kaede green was distributed throughout the connecting organoids, but Kaede red was almost absent (Fig. 4C). The central part of the axon bundle was irradiated with ultraviolet rays for 60 minutes to induce photoconversion. Furthermore, when the converted Kaede red protein was transferred into the cell over 2 hours (Fig. 4C), we succeeded in visualizing the axon bundle-related nerve cells as an organoid. The green and red distribution of Kaede revealed the location of neurons associated with axon bundles within the linked organoids (Fig. 4D). The red Kaede is similarly distributed on the left and right organoids (Fig. 4F). Within the organoid, it was found that there are more axon-related neurons in the region near the axon bundle (Fig. 4G). It was confirmed that the axons in the lowest layer of the connected organoids were distributed almost evenly over the entire z-axis, except that the axons in the lowest layer did not extend much into the bundle (Fig. 4H). Flow cytometry analysis of dissociated neurons of articulated organoids exposed to UV light revealed that from the Kaede green / red ratio (Fig. 4I), approximately 30% of neurons extended axons into the bundles between the organoids. It turned out that it was (Fig. 4J). In addition, the expression levels of FOS, TBR1, VGLUT1, GRIA2, and NEAT1 were significantly higher than those in which axon-related neurons were not related (Fig. 4K), which is the gene expression pattern of cluster 8 neurons in scRNA-seq. Was similar to (FIGS. 3C, G, H and 4L, M). From the two results of Kaede-labeling and scRNA-seq, it was derived that the neurons forming the axon bundle may be more mature neurons with high expression levels of TBR1, GRIA2, VGLUT1 and NEAT1. rice field. Nerve cells involved in the formation of this axon bundle may play an important role in performing advanced neural activity from an embryological and structural point of view (Fig. 4N).

2-5. Optigenic inhibition of axon bundles between connecting organoids To investigate whether axon connections play an important role in the complex neuronal activity of connecting organoids, neurotransmitter activity via axon bundles between organoids was conducted. An experiment was conducted to suppress it. Physical cleavage of the axon bundles between the organoids abolished burst-like neuronal activity and the LFP pattern in the δ wavelength band. Therefore, macroscopic connections are considered to be important for the complex and periodic neuronal activity patterns of brain organoids (Fig. 12).

To further investigate the role of axon connections between organoids, optogenic inhibition of axon bundles between organoids was performed. The microfluidic chip was modified to provide optogenic inhibition of axon bundles (Fig. 5A). ArchT (archrhodopsin; excretes H ⁺ extracellularly in response to light and suppresses neural activity) was expressed as an organoid using an adeno-associated virus (AAV) vector. Using an optical fiber and a PDMS lens, light suppressed only the axon bundles in the microchannel (Fig. 5B). Irradiation of axon bundles with orange light (568 nm, 20 ms, 20 Hz for 5 minutes) completely suppressed neuronal activity of each organoid, with highly amplified burst-like activity and low frequency under light irradiation. The δ-LFP pattern disappeared (FIGS. 5C and D). This result suggests that action potentials propagate through axon bundles and induce burst-like activity in connecting organoids. When the light irradiation was stopped, the burst-like activity and the δ-LFP pattern were immediately restored. The reaction induced by such light irradiation was repeatedly observed even after the light irradiation. The frequency of spontaneous burst-like activity did not change after light irradiation, suggesting that the unique properties of neural circuits within the organoid determine the frequency of spontaneous burst-like activity. ..

Also, the coherence (wave coherence) and synchronicity (synchronism) of the signals from each organoid of the connecting organoids were lost during the irradiation of light (FIGS. 5E and F). Optogenic disconnection of the connection between the two organoids significantly suppressed the overall intensity of neuronal activity, a phenomenon similar to that of patients with refractory epilepsy after surgery. Burst-like activity was abolished by optogenic inhibition of axons between organoids, but the number of action potentials observed was increased during light irradiation (FIGS. 5G and H). This result indicates that axons between organoids contribute to the induction of burst-like activity by harmonizing and integrating the activity of each neuron within the connected brain organoids. In addition, the brain hypothesis was reduced by light irradiation (Fig. 5I). This result indicates that the continuous firing of action potentials observed as a brain hypothesis is inhibited by blocking the axons between the organoids with light, and the connection between the organoids is important for the development of the brain hypothesis. ing.
These results indicate that activity transmitted via axon bundles between organoids underlies the complex neuronal activity that occurs in connecting organoids, demonstrating the importance of macroscopic connections in the brain. It is a thing.

2-6. Phase-Amplitude Coupling (PAC)
Culture of neural circuit tissue for more than 8 weeks further increased the LFP frequency and action potential spikes of the connecting organoids (Fig. 13A). In addition, the complexity of the signal has increased, and activity in the θ wavelength band has been frequently observed (Fig. 6A). In addition, a strong correlation was observed between the γ wavelength band and the δ wavelength band, and the γ wavelength band and the θ wavelength band. Therefore, in order to investigate the correlation between these wavelength bands, Phase-Amplitude Coupling (PAC), which is an index of the relationship between low-frequency activity and the amplitude of high-frequency spikes, was calculated. PAC is an established method for assessing the relationship between electroencephalograms (EEGs) in different frequency bands (Fell et al., Nat. Rev. Neurosci. 12 105-118 2011: Canolty et al., Trends Cogn. Sci. 14 506-515 2010). In the burst activity, the LFP waves in the δ wavelength band and the θ wavelength band appeared as synchronized burst activity in coordination with the γ wavelength band (Fig. 6B). The delta-γPAC modulation of the connecting organoid increased with continued culture, followed by an increase in theta-γPAC modulation (FIG. 6C). Both the δ-γ PAC modulation and the θ-γ PAC modulation that occur in the connected organoids were significantly higher than the PAC modulation in the single or fused organoids (Fig. 6D). Within each connected organoid, δ-γPAC and θ-γPAC showed a high degree of modulation (intraorganoid PAC). The δ-γ PAC modulation and θ-γ PAC modulation between organoids connected by axon bundles (modulation of interorganoid PAC) is higher than the PAC modulation within the organoid, which means that the δ and θ frequencies between the two organoids. It shows that the communication of the band is close (Fig. 6E).

Measurements by MEA have made it possible to record extracellular and local neuronal activity at high spatiotemporal resolution. However, the low density of electrodes compared to the cell density within organoids can make it difficult to understand the spike activity of action potentials from individual neurons. Ca ²⁺ imaging can measure with higher spatial resolution than the electrode-based method, but it is difficult to obtain high-magnification images from two organoids at the same time with a normal microscope. Therefore, we set up a microscope with two independent optics to simultaneously obtain individual Ca ²⁺ imaging of two organoids located a few millimeters apart. This system allows the acquisition of Ca ²⁺ imaging signals from two organoids with high spatiotemporal resolution, even when combined with MEA recording (Fig. 15A). The Ca ²⁺ indicator GCaMP6f was expressed in organoids connected by AAV for 3-7 days (Fig. 15A). The frames of the camera images of the left and right organoids were synchronized by a trigger signal, and the images were acquired at a speed of 20 frames per second. The change in fluorescence intensity (ΔF / F) in neurons in organoids was calculated. Fluorescent signals showed burst activity in both of the two organoids, synchronized with the activity of individual isolated neurons (Fig. 15B). The interval between bursts was consistent with the burst interval measured by MEA (Fig. 15C). Simultaneous measurements of MEA and Ca ²⁺ imaging showed a consistent increase in MEA signal and Ca ²⁺ concentration in organoids (Fig. 15D). To find a series of neurons with correlated activity, we created a correlation matrix of 26 neurons (13 neurons on the left organoid and 13 neurons on the right organoid, Figure 15F). During burst activity, neurons within the same organoid showed a strong correlation, but the correlation between neurons within different organoids was weak (Fig. 15G). When analyzing the correlation, when the neuronal activity within one organoid is shifted by 50 msec, a strong correlation between the organoids is observed, and thus there is a lag of 50 msec between the activities of the two organoids. Was consistent with the results observed by MEA (Fig. 1N).

Next, we investigated the neuronal avalanch of the signal recorded from the connecting organoid (Fig. 6F). Temporally proximal signals obtained from the electrodes were grouped and quantified as a brain hypothesis. The critical brain hypothesis index is considered a scale-free index of network critical dynamics (Beggs et al., Journal of Neuroscience 23 11167-11177 2003: Bowen et al., Frontiers in Systems Neuroscience 13 45 2019). The distribution of avalanche size increased with continued culture of connecting organoids. The index of the connected organoid brain hypothesis showed α = -2.8 5 weeks after culturing iPS cells (Fig. 6G). After that, it increased to α = -2.1 at 5.5 weeks and α = -1.6 at 8.5 weeks. This value of α theoretically corresponds to an index of -3/2 in the critical avalanche process. In addition, when the spatiotemporal pattern of the brain hypothesis was investigated by pattern recognition using the hidden Markov model, it was clarified that the variation of the pattern of the brain hypothesis increased depending on the culture time (). FIG. 6H).

2-7. Dynamic synaptic balance underlying complex neuronal activity of connecting organoids To examine the balance of synaptic receptors underlying complex neuronal activity of connecting organoids, connect organoids with synaptic channel antagonists CNQX, APV and vincurin. Processed. CNQX, APV and Bicuculline are antagonists of NMDA-type glutamate receptor (excitatory receptor), AMPA-type glutamate receptor (excitatory receptor) and GABA receptor (suppressive receptor), respectively (Figs. 6I-L, Figures). 13A and FIG. 14). No change in signal propagation rate was observed with antagonist treatment (FIGS. 13B and C). However, each antagonist affected neuronal activity such as action potential spikes and burst-like activity (FIGS. 6I and J). Since CNQX and APV reduced the neuronal activity of connecting organoids, namely δ-γ PAC and θ-γ PAC, it is the excitatory synapses that play an important role in inducing the complex activity of connecting organoids. It was suggested to be transmission (FIGS. 6K and L). On the other hand, bicuculline treatment significantly increased spike activity, but did not change the number of burst-like activities. However, burst-like activity was diminished by bicuculline treatment (Fig. 14). This result suggests that inhibitory synapses play an important role in producing cyclic neuronal activity of connecting connectoids.

Next, the effects of clinical compounds that change synaptic properties were investigated. Treatment with the GABA receptor agonists baclofen and diazepam reduced the number of spikes (FIGS. 6I and J). In addition, treatment with the schizophrenia drug Clozapine moderately reduced neuronal activity but did not affect PAC (FIGS. 6I, K and L). Treatment with the opioid buprenorphine specifically reduced theta-γ PAC, suggesting that buprenorphine affects the harmonization of activity in different frequency bands in connected organoids (Fig. 6I, FIG. K and L). These results indicate that the use of connecting organoids is effective in investigating the effects of compounds on complex neuronal activity.

2-8. Memory formation induced by optogenic stimuli in connected organoids To investigate the details of the response to external stimuli induced in connected organoids, optogenetic techniques were once again used. Channelrhodopsin was expressed in the connecting organoid to induce action potentials by light irradiation. Axons of connecting organoids that spontaneously ignite at 0.5 Hz were irradiated with pacing light (470 nm, 200 ms) at 0.5 Hz, the same frequency as spontaneous ignition, for 5 minutes. The connecting organoid was then stimulated at a higher frequency of 1.0 Hz for 5 minutes and then at 1.5 Hz for 5 minutes (Fig. 7A). Burst-like activity increased in line with the temporal stimulation pattern of light irradiation. When the stimulation was stopped, the frequency of burst-like activity was maintained at a high level for 10 minutes or more, and then returned to the frequency before the stimulation (FIGS. 7B and C). Thus, the sustained echo-like neuronal activity generated by photostimulation indicates that the temporal activity pattern of the connecting organoid is modulated by the external stimulus, and that the connecting organoid can maintain the temporal information of the stimulus. Furthermore, it suggests that connecting organoids can maintain temporal information as a primitive (basic) form of memory.
In particular, since there is a time lag between the start of stimulation and the occurrence of burst-like activity (Fig. 7C), it is suggested that multiple stimulations are required to modulate the activity of the connecting organoid. Also, during this time lag, the connecting organoid brain hypothesis expanded with periodic stimulation (Fig. 7D). This result indicates that the nerve cells that make up each organoid slowly adapt to the stimulus and establish a local circuit (subcircuit) that is easily excited by the time the activity of the connecting organoid activates in response to the temporal stimulus pattern. Suggest.

Stimulation of the connecting organoid at 1 Hz for 20 minutes induced sustained activity even after the stimulation was discontinued (FIGS. 7E and F). Burst activity continued to increase for approximately 20 minutes (decay period) after stimulation, after which burst activity returned to its original state. In contrast, the burst-like activity of the connecting organoids stimulated at the same frequency for 5 minutes immediately returned to the pre-stimulation state. Burst frequency increased during photostimulation, but no sustained increase in burst activity was observed. This result indicates that continuous stimulation is required to enhance circuit-level activity in connecting organoids.
Next, the effect of repeated periodic optogenic stimulation on connecting organoids was examined. Stimulation was performed 3 times every 60 minutes (FIGS. 7E and F). The burst frequency returned to normal from the stimulus-induced enhanced levels with each repeated stimulus.
In particular, when the stimulation time was 20 minutes, the delay time to respond to the external stimulus was significantly shorter for each second or third stimulus compared to the first stimulus (Fig. 7G). .. On the other hand, when the connecting organoid was stimulated for 5 minutes, the delay time to respond to the external stimulus did not change. These results suggest that the connecting organoid responds rapidly to the next repetitive stimulus through the network-level activity-enhancing effect of the previous stimulus.

Neuronal plasticity and memory are controlled by synaptic plasticity through a variety of molecular programs, including early and late responses, the calcium-dependent signal pathways required for each, and local protein synthesis. To explore the underlying mechanisms of memory formation in connecting organoids, the connecting organoids were treated with the CaM kinase II inhibitor K252a and the protein synthesis inhibitor anisomycin (Fig. 7H). Connected organoids responded to photostimulation and sustained post-stimulation activity was observed under K252a or anisomycin treatment. The duration of sustained activity decay after stimulation was significantly reduced in the presence of K252a after the second and third stimulations (Fig. 7I). In contrast, anisomycin treatment resulted in a slight reduction in sustained activity only after the third stimulus. In addition, compared with the control treatment, the K252a treatment inhibited the shortening of the time lag from the start of the second stimulation to the response (Fig. 7J).
These results suggest that the calcium-dependent signal pathway supports the short-term potentiation observed in connected organoids.

The brain hypothesis was also reduced by K252a treatment (Fig. 7K). The number of hidden patterns in the brain hypothesis during light irradiation decreased compared to the nerve avalanche during non-light irradiation (FIG. 7L). This result suggests that during light irradiation, the activity of organoids is immobilized by a stimulus of a certain cycle, and as a result, a brain avalanche is less likely to occur and the amount of information possessed by the neural activity is reduced. In particular, the number of hidden patterns gradually increased with each stimulus, suggesting that the neural circuits within the connecting organoids developed and matured due to the stimuli and the proliferation of neural activity by the stimuli. do. The anisomycin treatment suppressed the expansion of the hidden pattern of the brain hypothesis during the light irradiation discontinuation period (Fig. 4M). Therefore, although the conventional long-term potentiation form was not observed, the long-term potentiation (Fig. 4M) was observed. It is suggested that the mechanism of long-term potentiation) was partially activated in the connecting organoids.

Next, in order to investigate the complexity of the activity of connecting organoids, Higuchi's fractal dimension was calculated. The fractal dimension (FD) is an indicator of the complexity of fine structures and has been used to assess the complexity and temporal changes of EEG signals (Varley et al., PLOS ONE 15 e0223812 2020). In the control, after the first (1st) photostimulation was stopped, the FD increased and the increased FD was maintained even in the absence of the second or third light irradiation (FIGS. 7N and O). This result suggests that the entire network gained complexity on the first stimulus and remained complex thereafter. In the presence of K2532a, FD did not increase with the first light stimulus, but increased with the second light irradiation, and this FD increase was maintained by the third light irradiation. This result suggests that K252a treatment disrupted network activity enhancement and complexity. In contrast, in the presence of anisomycin, FD did not change with light stimulation.

In order to deepen the understanding of burst-like activities, the induced activities were sorted and compared (Fig. 8A). Detailed analysis revealed that photostimulation induces multiple brain waves of neuronal activity in burst-like activity. In control, the time lag of burst-like activity became shorter with each repeated stimulus (Fig. 8B). This reduction in time lag was also observed under anisomycin treatment, but not in the presence of K252a. To analyze the induced burst-like activity in more detail, the probabilities of the induced bursts were calculated (FIGS. 8C and D, kernel density estimation). As a result, it was found that a second-order response and a third-order response occur after the sharp first peak. At the first stimulus, the first peak was observed near the end of the stimulus and a weak secondary peak was observed. With the second and third stimuli, the peak of the first response became stronger and shifted towards the onset of stimuli. In addition, the secondary and tertiary peaks became sharper and stronger with each repeated stimulus (FIGS. 8C and D). Second- and third-order waves were also observed under K252a and anisomycin treatment. In particular, the left and right organoids connected via axon bundles sometimes responded to light irradiation with slightly deviated kinetics (Fig. 8E). Also, the second and third waves of the induced burst response were observed alternately between the two connected organoids (Fig. 8E). These results suggest that activity in connected organoids is produced and developed by a complex combination of activity within and between the left and right organoids.

Next, in order to quantify the various patterns of induced activity, the entropy of the burst-like activity waveform obtained from the connected organoids was calculated (Fig. 8F). Repeated stimulation increased the entropy of induced burst-like activity, indicating that this quantification method can capture the development of complex burst waves. This increase in entropy was inhibited by K252a treatment but not by anisomycin treatment. Therefore, it was suggested that the change of burst wave is also promoted by the mechanism of calcium signaling dependence.

The present invention provides in vitro neural circuit tissues that exhibit complex activity and plasticity, especially neural tissues that mimic brain function. In addition, an observation device for the nerve activity and a method for screening a substance using the device are provided. Therefore, it is highly expected to be used as a brain model in the medical field, pharmacy field, and the like.

Claims (36)

Neural circuit tissue induced in vitro, in which two or more nerve cell clusters are connected via axons. The neural circuit tissue according to claim 1, wherein the nerve cell mass is an organoid. The neural circuit tissue according to claim 1 or 2, wherein the two or more nerve cell clusters exhibit activities related to each other. The neural circuit tissue according to claim 3, wherein the interrelated activities occur with a time difference of 500 milliseconds or less. The neural circuit tissue according to claim 3 or 4, wherein the interrelated activities are synchronized activities. The neural circuit tissue according to any one of claims 1 to 5, which performs spontaneous ignition activity. The neural circuit tissue according to claim 6, wherein the spontaneous firing activity is 50 times or more per minute. The neural circuit tissue according to any one of claims 1 to 5, which performs burst activity. The neural circuit tissue according to claim 8, wherein the coefficient of variation of the frequency of the burst activity is 0.2 or more. The neural circuit tissue according to any one of claims 3 to 5, wherein the activity of each nerve cell mass connected via the axon indicates coherence. The neural circuit tissue according to claim 10, wherein the interference differs for each frequency band. From claim 1, the local field potential detected from the nerve cell mass includes a δ wavelength band (0.5 Hz-4.0 HZ) component and / or a γ wavelength band (300 Hz-3000 Hz) component. The neural circuit tissue according to any one of claims 11. The neural circuit tissue according to claim 12, wherein the local field potential further contains a θ wavelength band (4.0 Hz-8.0 Hz) component. The neural circuit tissue according to any one of claims 1 to 13, wherein the nerve cell mass is induced to differentiate from pluripotent stem cells. The neural circuit tissue according to claim 14, wherein the nerve cell mass is formed by culturing pluripotent stem cells for 6 weeks or more. The neural circuit tissue according to claim 14 or 15, wherein the pluripotent stem cell is an iPS cell (induced pluripotent stem cell). The neural circuit tissue according to any one of claims 1 to 16, which is obtained by culturing the nerve cell mass for 2 weeks or more. The neural circuit tissue according to any one of claims 1 to 17, which exhibits plasticity to external stimuli. An in vitro induced neural activity observer in which two or more nerve cell clusters are connected via axons.
A substrate, a plurality of wells provided on the surface of the substrate for accommodating the nerve cell mass, and the well for guiding and extending the axon so as to connect the nerve cell mass to each other. Including guide grooves provided by connecting,
An apparatus for observing neural activity of neural circuit tissue, characterized in that an electrode is provided in each of the wells, and an analysis unit for analyzing the correlation of electrical signals from the electrodes is further included. The device for observing neural activity of neural circuit tissue according to claim 19, wherein the analysis unit separates each of the electrical signals for each frequency band and analyzes the correlation by phase amplitude coupling. The device for observing neural activity of neural circuit tissue according to claim 19, wherein the analysis unit analyzes the correlation by wavelet coherence. The analysis unit classifies the nerve activity into any one of action potential, burst activity, brain hypothesis, and local field potential, according to any one of claims 19 to 21. A device for observing neural activity of the described neural circuit tissues. The device for observing neural activity of neural circuit tissue according to claim 22, wherein the analysis unit has the signal pattern corresponding to the neural activity in advance and classifies the neural activity by collating with the signal pattern. The device for observing neural activity of neural circuit tissue according to claim 23, wherein the neural cell mass and / or the axon is externally stimulated to form the neural activity, and the corresponding signal pattern is acquired in advance. .. The device for observing neural activity of neural circuit tissue according to claim 2, wherein the stimulus is any one or more of light irradiation, electrical stimulation, or compound administration. The device for observing neural activity of neural circuit tissue according to claim 25, wherein the stimulus enhances or suppresses the neural activity. The device for observing neural activity of neural circuit tissue according to any one of claims 19 to 26, wherein the electrodes are formed by arranging a plurality of electrodes in an array at the bottom of the well. The device for observing nerve activity of neural circuit tissue according to claim 27, wherein the electrode receives the electrical signal from the nerve cell mass and electrically stimulates the nerve cell mass. The device for observing neural activity of neural circuit tissue according to claim 27 or 28, wherein the substrate is transparent so that the nerve cell mass in the well can be optically observed from the bottom. 19. Any one of claims 19 to 29, wherein a light irradiation device for optically stimulating the nerve cell mass and / or the axon is provided so as to face the surface of the substrate. A device for observing neural activity of the described neural circuit tissues. The device for observing neural activity of neural circuit tissue according to claim 30, wherein the light irradiation device includes an irradiation unit that locally applies light irradiation to the nerve cell mass and / or a part of the axon. An in vitro induced neural activity observer in which two or more nerve cell clusters are connected via axons.
The substrate, a plurality of wells provided on the surface of the substrate for accommodating the nerve cell mass, and the well for guiding and extending the axon so as to connect the nerve cell mass are connected. Including the guide groove provided in the
Each of the wells is provided with an optical system for measuring the light intensity from the nerve cell mass, and further includes an analysis unit for acquiring the correlation of the light intensity signal from each of the nerve cell clusters in the well. A device for observing neural activity of neural circuit tissues. Two of the wells are provided to the substrate, the first objective lens of the optical system is brought close to one of the wells from one surface of the substrate, and the second objective lens is from the other surface of the substrate. The device for observing neural activity of neural circuit tissue according to claim 32, which is provided close to the other of the wells. The device for observing neural activity of neural circuit tissue according to claim 32 or 33, which detects light emission from a calcium fluorescent probe and detects an increase in calcium ion concentration in the nerve cell mass caused by an action potential. The device for observing neural activity of neural circuit tissue according to claim 34, wherein a light beam for causing the light emission is incident from a side end surface of the substrate. A method for screening a substance that changes the neural activity of neural circuit tissue by using the observation device according to any one of claims 19 to 35.
A substance screening method in which a target neural circuit tissue is set in the observation device, the substance is given to the neural circuit tissue, and fluctuations in the neural activity are observed.

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