JP2005147686A - Apparatus for collecting intracerebral substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for collecting an intracerebral substance, analyzing the collected intracerebral substance, and giving new knowledge regarding the plasticity of the brain and a neural network system. <P>SOLUTION: The apparatus for collecting the intracerebral substance comprises a stimulus means for intending to give a stimulus to a nerve nucleus at the intracerebral upstream; and a means for collecting the intracerebral substance intended to be mounted to a plurality of nerve nuclei at the downstream existing in a stimulus propagation path for propagating a stimulus by the stimulus means. Additionally, a model mouse comprises a stimulus means for giving a stimulus to the nerve nucleus at the intracerebral upstream; and a means for collecting the intracerebral substance to be mounted to the plurality of nerve nuclei at the downstream existing in the stimulus propagation path for propagating a stimulus by the stimulus means. Further, a method for analyzing a signal transmission system substance uses the model mouse. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a brain substance collection apparatus and model mouse, and a method for analyzing a signal transduction substance using the model mouse, and more particularly, a brain substance collection apparatus and model mouse using stimulation to the nerve nucleus, and The present invention relates to a method for analyzing a signal transduction substance using the model mouse.

  Neural networks are broadly divided into central nerves that issue various commands and peripheral nerves that transmit them. Nervous cells can be divided into neurons (nerve cells) and glial cells that help the neurons. Neurons communicate with each other through a junction called a synapse. One neuron receives information from more than several thousand synapses as a neurotransmitter that is a chemical signal, and receives that information through ion channels in the cell body and dendrites. As a result, a change in membrane potential occurs in the neuron, and the generated action potential is sent along the axon to the nerve terminal. Many molecules are involved here, and their interaction causes a change in information transmission efficiency (plasticity). High-order functions such as learning, memory, and thinking are exhibited by the function of such neurons.

  The central nervous system includes the brain and spinal cord. Among these, the brain is composed of a complex neural network, adapting to changes in the outside world, and not only functions such as blood circulation and breathing and instinctive needs, but also recognition, motor control, consciousness, It manages all of the sympathy such as emotions and higher-order functions such as memory learning.

  For example, taking a basic process at the cellular level of learning and memory as an example, modification and new generation of a neural circuit accompanying neuroplasticity are known. This is because long-lasting and persistent nerve plasticity changes, so that nerve cells newly have neural communication, and change their existing communication pathways, exhibiting coordinated function expression As a result, the memory learning ability is acquired.

  In recent years, it has been found that neural stem cells also exist in the completed adult brain, and that new neurons are born. On the other hand, most neurons in the brain are already determined after birth, and neuroplasticity is helpful in recovering from brain damage and degenerative diseases. Also, if the plastic response is excessive or goes out of control, it causes brain functional disorders such as epilepsy and stress disorders.

  Thus, when the plastic change of the nerve is seen at the molecular level, it is considered that various intracellular and external information transmission mechanisms are involved.

In recent years, the signal transduction site between T cells and antigen-presenting cells has been called immunological synapse because its function is similar to that of neuronal synapses. On the other hand, lectins have been substituted for lymphocyte activation for a long time, suggesting the action of sugar chain molecules in immunologizal synapse. According to recent findings, the addition of CD8 and CD43 sialic acid by ST3Gal I is deeply involved in the antigen recognition ability of CD8 + T cells, activation and differentiation into memory T cells, and this phenomenon is the expression of ST3Gal I It has been shown to depend on a decrease in the amount (Cell (2001) 107: 501, Immunity (2000) 12: 273).
Cell (2001) 107: 501, Immunity (2000) 12: 273

  However, the above-described method for recovering brain substances cannot be recovered from a plurality of nerve nuclei corresponding to the stimulus propagation path. Moreover, it was considered impossible to use a mouse, which is a small animal.

  Also, because of the structural features of the brain, that is, the brain is composed of complex neural networks and is composed of heterogeneous populations such as neurons and glial cells that have different functions even in specific nerve regions. Therefore, there are almost no reports on the function expression mechanism and information transmission mechanism at the molecular level.

  If it is possible to collect and analyze substances from the above-mentioned nerve nuclei, it will be possible to discover new knowledge about brain plasticity and to obtain more information about the mechanisms of the neural circuit system. .

  Accordingly, an object of the present invention is to provide an apparatus and an analysis method that can collect a substance in the brain, further analyze the collected substance in the brain, and provide new knowledge about the plasticity of the brain and the neural circuit system.

  In order to achieve the above object, the inventors focused on the importance of the nucleus in the propagation path of the stimulus in a series of neural circuit systems, and as a result of earnest research using model mice, It came to find the internal substance collection | recovery apparatus, the model mouse, and the analysis method of the information transmission substance using the said model mouse.

  According to the apparatus of the present invention, there is an advantageous effect that information generated in a nerve nucleus existing in a series of stimulus propagation paths can be continuously grasped.

  In addition, the model mouse of the present invention can collect brain substances corresponding to the stimulus from a plurality of nerve nuclei in the stimulus propagation path. Thereby, there is an advantageous effect that an algorithm of a substance change in the stimulus propagation path and a substance transport algorithm in the path can be analyzed in a specific neural circuit.

  Moreover, according to this invention, there exists an advantageous effect that it can be made useful for discovery of the novel brain substance.

  Moreover, according to this invention, there exists an advantageous effect that a chemical | medical agent effect can be investigated in multiple places at the time of a stimulus response.

The intracerebral substance collection device of the present invention comprises a stimulation means intended to stimulate an upstream nucleus in the brain, and a plurality of downstream nerves present in a stimulation propagation path for transmitting the stimulation by the stimulation means. And means for collecting substances in the brain intended to be attached to the nucleus. Here, upstream means mainly the source of information flowing in the nervous system. For example, it can be said that it is a stimulated part. Downstream mainly means the side that receives the source from which information is sent, the so-called information source. Using the limbic system composed of the amygdala-hippocampus-thalamus-cerebral cortex, when explained, when the stimulus is applied to the amygdala, the amygdala side means upstream, while the cerebral cortex side means downstream To do.
The stimulating means is not particularly limited as long as it provides a stimulus for viewing the information flow of the neural circuit and can provide a stimulus to the upstream nerve nucleus. Examples of such stimulation include electrical stimulation, drug administration to a limited site, limited neural circuit disconnection, learning, and the like. As stimulation, it is preferable to use electrical stimulation.

  Various electrodes can be used for the electrical stimulation and are not particularly limited. For example, a microporous electrode or the like can be used with tungsten, sintered titanium, sintered titanium nitride, and microporous carbon or graphite. The electrode may be polished or the like to increase the surface area. In addition, a porous material using titanium, platinum, iridium or the like may be used. In short, the stimulating means should be used by appropriately changing it according to various circumstances such as the target brain substance, neural network, etc., and the above-mentioned in the present invention, or other materials depending on necessity Stimulation electrodes can be used. However, when installed in an animal, a tungsten wire can be preferably used from the viewpoint of preventing deterioration.

  The brain substance recovery means is attached to a plurality of downstream nerve nuclei existing in the stimulus propagation path for transmitting the stimulus by the stimulus means. This device not only knows how the information from the upstream nucleus is propagated and gives instructions to the downstream nucleus, but also the mechanism of the plasticity of the brain and the various types of plasticity involved. It is also possible to grasp new information transmission substances. It is also possible to elucidate the causes of various diseases based on the knowledge of such novel information transmission substances.

  The neural circuit applicable to the present invention is not particularly limited, and can be applied to various neural circuit systems. For example, as the neural circuit system, the amygdala-hippocampus-thalamus-cerebral cortex limbic system is cited in the following examples, but in addition, the perceptual system including the visual system, the auditory system, and the motor system And so on.

  Next, the model mouse of the present invention will be described. The model mouse of the present invention is attached to a stimulation means intended to stimulate an upstream nucleus in the brain, and a plurality of downstream nerve nuclei existing in a stimulation propagation path for transmitting the stimulation by the stimulation means. And means for collecting substances in the brain that are intended.

  The model mouse according to the present invention is not particularly limited as long as it has the above-described stimulation means and brain substance collection means. Depending on the purpose, as well as normal mice (wild-type mice that have not been treated at all), after preparing knockout mice, knockin mice, and transgenic mice, stimulation means and brain substance collection means may be installed. . Moreover, these are not limited to mice, but can be applied to other animals.

  In the present invention, an animal such as the mouse can also be used to analyze information transmitters in nerve cells. The analysis method of the present invention uses animals. Examples of such information transmitting substances include acetylcholine, catecholamine, serotonin, histamine, and adenine nucleotide, as well as amino acids such as glutamic acid, GABA, and glycine.

  For example, when visualizing the expression of a specific gene, the thalamic nerve cell, and the nerve communication pathway for the purpose of elucidation at the molecular level between cells and in the extracellular environment, an animal such as a wild type mouse or the like It can be carried out using an animal such as the above-mentioned mouse in which a specific gene is modified. In the present invention, it is possible to analyze information-transmitting substances derived from nerve cells, glial cells, vascular endothelial cells, choroidal endothelial cells, and the like using animals such as the aforementioned mice. For example, the analysis of intracellular signaling substances can be performed by analyzing multiple downstream nerve nuclei and nearby brain substances that exist in the path of transmission of stimuli to the upstream nerve nuclei in the brain. Can be done.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not intended to be interpreted as being limited to the following examples.

Example 1
First, in order to elucidate the mechanism in the neural circuit system at the molecular level, we investigated the information transmission mechanism using mice as model animals.

  Glycosylation is involved in various vital functions, especially regarding immune system and cancer metastasis, elucidation of the mechanism by which sugar chain molecule recognition and response signals between cells and in the extracellular environment are transmitted to the intracellular signal transduction system. Because of the progress, we focused on sugar chain molecules. The sugar chain structure is quite different between lower animals and mammals including humans. For example, flies and nematodes are effective for analysis of a series of individual, cell, and molecular levels, but have a feature that they hardly have sialic acid.

  In addition, the induction of development / differentiation and canceration is accompanied by fluctuations in various gene expressions, but in recent years, examples have been given in which sugar chains are involved in the initial stages of signal transduction systems related to life phenomena. For example, the Notch signal plays an important role in determining cell fate. This Notch signal begins with the addition of acetylglucosamine to Notch O-fucose by Fringe, a glycosyltransferase. This Notch signal is strictly governed by the fringe expression location and timing, and then many factors (Notch itself, Delta, Serrate, Wingless, etc.) are induced and suppressed (Segmentation Clock). It is also known that CD43, which is a substrate molecule of ST3Gal I, and moesin that binds to CD43 exhibit expression changes with T cell activation and canceration. On the other hand, PDZ domain protein CASK that binds to proteoglycan syndecan-2 is also bound to the NMDA receptor via Lin in the nervous system. It has been suggested that CASK may enter the nucleus together with T-box transcription factors to regulate gene expression. In addition, it has been reported that proteoglycans such as glypican and biglycan, which are localized on the nerve cell membrane of the mature brain, are directly transferred to the nucleus. However, in the nervous system, there is no knowledge that links sugar chain modification and signal transduction system. There is a high possibility that there is a signal transduction system related to ST3Gal IV that shows an increase in expression linked to nerve plastic changes, and the presence of a factor that shows expression induction or suppression linked to ST3Gal IV expression is expected.

  In addition, sugar chain molecules are synthesized by cooperation of a series of glycosyltransferase groups. Therefore, functional expression via sugar chain molecules is considered to be governed by the location and timing of glycosyltransferase expression. In addition, regarding the experimental system in the actual immune system and cancer metastasis, the same type of immunocompetent cells and cancer cells can be grown in a culture system (in vitro) and then returned to the animal body (in vivo). Since it is possible, analysis from the individual level to the cell / molecular level is possible.

  From the complex structural features of the brain: 1. Visualization of nerve cells expressing certain sugar chain functional molecules and their nerve connections in the brain (in vivo). 2. Separation of visualized neuronal population in culture system By enabling observation of candidate molecules that can participate in the signal transduction system via sugar chains in neurons visualized in the brain and culture systems, information transmission mechanisms at the individual, cell, and molecular levels in the nervous system are also possible. It is expected that this analysis will be possible.

  The inventors have: 1. 1. α2,3-sialyltransferase (ST3Gal IV) is expressed only in 2% thalamic neurons, subcerebellar granule cell layer, and single granule cells in the mature mouse brain. 2. Thalamic expression shows an increase with progression of the kindling stage, and 21% of thalamic neurons show its expression at the final stage when neuroplasticity is acquired. This increase in expression is not observed in other existing α2,3-sialyltransferases. 4. By two-dimensional electrophoresis, the two glycoproteins that can serve as ST3Gal IV substrates are It has been clarified that the cerebral cortex) shows 46-fold and 60-fold increased expression.

  The thalamus receives a lot of information from each brain area of the sensory system, motor system, and limbic system, and propagates the information to the cerebral cortex. The loss of function due to tumor and nutritional vascular disorders also reveals an important brain relay / integration center. Since ST3Gal IV shows expression restricted to this thalamic neuron and a marked increase in expression in response to stimulation, the glycan molecule that undergoes sialic acid addition by ST3Gal IV contributes to the functional expression of the thalamus and relay nucleus The potential for contribution is expected to be high.

  FIG. 1 shows a dentate cross-sectional view of a mouse and shows nerve plasticity in kindling mice. From the left, the behavior, brain faction, gene expression, and micromorphology are shown. In the fine form of the photograph on the right, Sham shows the control and Kindling shows the kindling treatment. Dendraite indicates dendrites. Dendritic development was observed in the kindling treatment.

  A specific outline is shown in FIG. 3 and FIG. FIG. 3 shows the preparation of kindling mice. In the figure, Complex Neural Cicuit represents a complex neural circuit. Kindling amygdala was used as the circuit extract. Hip indicates the hippocampus, Thalamus indicates the thalamus, and Cingulate indicates the cerebral cortex. FIG. 5 is a diagram showing an embodiment when stimulating a mouse. As shown in the photograph of FIG. 5, a guide or the like can be fixed by a probe for applying a stimulus and a fixing means (cement) for fixing the probe. In order to embed multiple microdialysis probes for mice in the brain, the guide is not closed with screws, and the guide and dummy are fixed. Actually remove the dummy the day before the substance collection date in the brain and fill the probe. This time, the guide diameter is 0.3mm.

  From this device, brain substances were collected from three nerve nuclei located in the stimulus propagation path, and brain fluid was also collected. In addition, by collecting the intraventricular fluid, it was possible to study molecules with a large molecular weight. This time, it was possible to determine whether known glutamate, glycine, and GABA exist in each nucleus and how they change in response to stimulation.

  In addition, as a change to the neural network system, it can be seen that the development of Dendlite (dendrites) is remarkable in kindling. Since glycan molecules are synthesized by the cooperation of a series of glycosyltransferase groups, functional expression via glycan molecules is considered to be governed by the location and timing of glycosyltransferase expression.

  In addition, nerve plasticity in kindling mice was also examined. The results are shown in FIG. From left to right, behavior, brain waves, gene expression, and fine morphology are shown. In the fine form of the photograph on the right, Sham shows the control and Kindling shows the kindling treatment. It can be seen that ST3Gal IV signal enhancement in the thalamus is linked to behavioral changes and brain wave transitions. As a result, it was found that the formation of dendrites is particularly remarkable in the process of plasticity of the neural circuit by applying a stimulus to a specific neural circuit system.

  FIG. 6 shows the results of actually examining the effects on stimulation and the neural circuit system using the apparatus. FIG. 6 shows the increase in dendrite width after kindling. In FIG. 6A, Molecular layer indicates a molecular layer, and Granular Layer indicates a granular layer. (Hylus indicates polymorphic cells. The left picture shows the kindling treated (Kindling) and the right picture shows the control (Sham). FIG. 6 (b) shows the kindling treated and control cases. Comparison of dendrites is shown.

  FIG. 2 shows an illustration of a neural circuit system applicable to the brain substance recovery apparatus of the present invention. The effects of glycans on the plasma membrane were clarified as a result of investigating the effects and effects of brain substances on the hippocampus, thalamus, and cerebral cortex on the propagation pathway by kindling stimulation of the amygdala.

  It was possible to further investigate the neuronal plasticity in nerve cells that occurs when a stimulus is applied in a specific neural circuit. In the following, we investigated the effects of dendritic abnormalities and the neuroplasticity of sialylation of glycoproteins and glycolipids.

  First, we investigated the abnormalities of dendrites in the hippocampal dentate gyrus and granule cell layer and amygdala hippocampal transition area after acquiring epilepsy by kindling.

  Amygdala kindling is a model of human temporal lobe epilepsy. To explain this, when a slight electrical stimulation was applied once a day, animals that initially showed only a small partial seizure response increased the weight of the seizure symptoms and prolonged afterdischarge as the number of stimulations increased. I finally got a generalized seizure. The appearance of post-launch is important for the kindling formation, not the electrical stimulation itself. Also, post-fires in this limbic system are acquired permanently. In this system, the inventors examined dendritic changes in mice after acquiring seizures induced by kindling. First, immunohistochemical staining of the hippocampus and amygdala on the side opposite to the electrode insertion side was performed by a fluorescent antibody method using an anti-MAP-2 antibody. MAP2 is a marker of neuronal cell bodies and dendrites. As a result of staining, after kindling acquisition, the hippocampal dentate gyrus / granule cells and neurons in the amygdala hippocampal transition area showed enlargement of the proximal dendrites. In order to analyze the dendritic morphological changes in more detail, the side opposite to the stimulus was observed at the electron microscope level. As a result, in the hippocampal dentate gyrus / granule cells and neurons of the amygdala hippocampal transition area, after the kindling was acquired, the number of dendrites including polymerized microtubules increased, and the cross-section of the dendrites was enlarged. In addition, bundling between dendrites was observed only in hippocampal dentate gyrus and granule cells, and puncta adhaerentia was observed between dendrites. Furthermore, the enlargement of dendrites in the hippocampal dentate gyrus was more pronounced in the granule cells on the rostral side than on the caudal side. These results revealed that microtubule-rich dendritic hypertrophy occurs as part of the structural plasticity associated with epileptic seizures in certain areas of the mature brain.

  In this way, it has become possible to detect brain substances and analyze nerve cells using the model mouse of the present invention.

Next, the expression variation of the induced sialyltransferase mRNA was also examined in the mouse hippocampus after acquiring epilepsy by kindling.
Sialic acid plays an important role in various life phenomena, and there are several examples suggesting that sialylation of glycoproteins and glycolipids affects neuroplasticity in the brain.

  There are 19 types of sialyltransferases that sialylate into sugar chain structures, but some of them have overlapping substrate specificities. Therefore, the expression distribution of sialyltransferase is an important factor for adding sialic acid to a specific sugar chain structure.

  In fact, when RT-PCR was used to screen sialyltransferases expressed in the mature brain and hippocampus, seven sialyltransferases: ST3Gal I-IV, ST8Sia IV, ST65Gal I, and ST6GalNAc II were expressed in the hippocampus. I understood. In addition, in situ hybridization histochemistry suggested that each cell expressed an appropriate sialyltransferase. Furthermore, the expression of several sialyltransferases in the hippocampus due to amygdala-kindling showed expression fluctuations linked to neuroplasticity. For example, ST3Gal IV and ST6GalNAc II mRNAs increased expression, ST3Gal I and ST8Sia IV decreased expression, and other ST3Gal II, ST3Gal III, and ST6Gal I did not change. These results suggest that the expression of sialyltransferase is controlled by physiological activity and is deeply involved in neuroplasticity.

  Next, the region-specific and epilepsy induction-dependent expression of six α2,3-sialyltransferases (ST3Gal I-VI) was also examined in the mature mouse brain.

  The sialylated glycan structure plays an important role in the expression of various biological functions, and the sialylated glycan structure is also observed in the brain, and sialic acid addition has some effect on neuroplasticity. The possibility of giving was proposed. However, in order to clarify the role of sialic acid addition in the brain, it was necessary to first determine the nerve cells exhibiting sialic acid addition.

  The inventor first clarified the distribution of α2,3-sialyltransferase (ST3Gal I-VI) mRNAs in the brain by in situ hybridization histochemistry. The distribution in the brain was compared with and without physiological stimulation. First of all, each transferase showed a region-specific expression distribution. ST3Gal II, III, V mRNAs were expressed in neurons in the brain. On the other hand, ST3Gal I, IV and VI mRNAs were expressed only in limited brain regions. Next, in order to examine whether the mRNA expression of these six sialyltransferases is stimuli responsive, the inventors examined the effect of kindling on epilepsy induction. As a result, the increase in the expression level of ST3Gal IV in the thalamus was particularly remarkable. In the prethalamic nucleus, the number of ST3Gal IV-expressing neurons increased to 2-21% as the stage of epilepsy acquisition progressed. Furthermore, the final product of ST3Gal IV was still significantly increased after kindling by Western blotting and immunohistochemistry. These results provide a molecular biological basis that reveals when and where sialylated glycan structures function with neuroplasticity.

  According to the present invention, in the field of bioscience, it can be used for basic research of brain science in general, and can be greatly used for drug discovery testing and pharmaceutical systems.

FIG. 1 shows nerve plasticity in kindling mice. From the left, the behavior, brain faction, gene expression, and micromorphology are shown. In the fine form of the photograph on the right, Sham shows the control and Kindling shows the kindling treatment. It can be seen that ST3Gal IV signal enhancement in the thalamus is linked to behavioral changes and brain wave transitions. FIG. 2 shows an illustration of a neural circuit system to which the present invention can be applied. In the figure, 1) Complex Nneural Cicuit indicates a complex neural circuit. Kindling amygdala was used as the circuit extract. Hip indicates the hippocampus, Thalamus indicates the thalamus, and Cingulate indicates the cerebral cortex. 2) Orchestration of neural connection shows the organization of neural connections. 3) The Contribution of Glycan on Plasmaembrane shows the function of glycans on the plasma membrane. FIG. 3 shows the preparation of kindling mice. In the figure, Complex Nneural Cicuit indicates a complex neural circuit. Kindling amygdala was used as the circuit extract. Hip indicates the hippocampus, Thalamus indicates the thalamus, and Cingulate indicates the cerebral cortex gyrus. FIG. 4 shows an outline of a brain substance recovery apparatus according to an embodiment of the present invention. Kindling Stim. Is an abbreviation for kindling stimulation. Micro-dialysis is an abbreviation for microdialysis. FIG. 5 is a diagram showing an embodiment when stimulating a mouse. FIG. 6 shows the increase in dendrite width after kindling. In FIG. 6A, Molecular layer indicates a molecular layer, and Granular Layer indicates a granular layer. Hylus refers to polymorphic cells. The left picture shows the kindling treated (Kindling), and the right picture shows the control (Sham). FIG. 6B shows a comparison of dendrites between the kindling treatment and the control.

Claims (7)

  Stimulation means intended to stimulate the upstream nucleus in the brain, and collection of substances in the brain intended to be attached to a plurality of downstream nerve nuclei existing in the stimulus propagation path for transmitting the stimulation by the stimulation means A device for collecting substances in the brain.   2. The apparatus according to claim 1, wherein the stimulation means is based on electrical stimulation.   The device according to claim 1 or 2, wherein the upstream nucleus is the amygdala.   The device according to claim 1, wherein the plurality of downstream nerve nuclei are a hippocampus, a thalamus, and a cerebral cortex.   Stimulating means intended to stimulate the upstream nucleus in the brain, and a substance in the brain intended to be attached to a plurality of downstream nuclei existing in the stimulus propagation path for transmitting the stimulation by the stimulating means A recovery mouse, and a model mouse.   6. A method for analyzing a signaling substance in nerve cells using the model mouse according to claim 5.   The method according to claim 6, wherein analysis of a signal transduction system substance in a nerve cell is performed by analyzing the downstream nerve nucleus and a substance in the vicinity of the brain.

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