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Exploring the structure and mechanism of the cerebral cortex
RIKEN Brain Science Institute |
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| Discovery of the honeycomb structure | |||
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It was when we were examining under a microscope the distribution of the calcium-binding protein parvalbumin in a thin slice of the temporal lobe, part of the cerebral cortex, of a monkey. "Kathy was the first to find it. I remember her saying, ‘I see something in here.'" Noritaka Ichinohe, a research scientist at the Laboratory for Cortical Organization and Systematics recalled the moment of the discovery. Kathleen S. Rockland, Laboratory Head, is called Kathy by her laboratory members. Ichinohe and other research scientists in the laboratory continued the examination using different staining methods, through which they successfully confirmed the existence of the honeycomb structure in the surface layer of the cerebral cortex. This structure was observed over quite an extensive area of the cerebral cortex of the monkey. The researchers later found the structure also in the cortex of mice, rats, and humans. Ichinohe says, "The honeycomb structure must be playing an important role in the functioning of the cerebral cortex since it is observed in different species." A neuron is a cell body with specializations. It generates electrical signals, which are transmitted to other neurons through an axon, a long projection dividing into many branches towards its end. The cell body also has many branches called dendrites which receive signals from other neurons. The cerebral cortex, which is about 2.5 mm thick, is the outer surface of the cerebrum. The cortex, a collection of neurons approximately similar to each other in function and form, consists of six layers (Figure 1). Layer I is the outermost layer, which is filled with branched axons and dendrites. Under Layer I is Layer II consisting of smaller neurons. The honeycomb structure which has been discovered by Rockland and her laboratory members lies between Layers I and II. Figure 2 shows a stained, vertical section of the boundary of the two layers. The areas stained in green are dendrites arising from the deeper layers of the brain. You can see in the image that many of them gather to form a bunch. The red areas represent parvalbumin, showing that there are other bunches of dendrites arising from the shallower layers. We assume that a different bunch of dendrites serves as an intake for different information. Figure 3 is a horizontal section of the same boundary stained using a different method. The areas stained in green and red in Figure 2 correspond to those areas in white and brown in Figure 3, respectively. The image shows a honeycomb-shaped structure. This honeycomb structure is considered a new type of "column" that reveals new aspects of the conventional theory.
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| Exploring unknown functions of the cerebral cortex | |||
 A column is a functional unit running vertically in the cerebral cortex. Sizes vary according to their location but generally about 0.5 mm in diameter. Visual information that enters the eyes, for example, will be sent to the thalamus at the back of the brain, which then transmits the information to the primary visual cortex (V1) in the occipital region. In this area, basic information about shapes, colors, and movements is further analysed. While some columns in V1 consist of neurons that only react to lines with a certain slope, neighboring columns may consist of neurons reacting to lines with a different slope. V1 projects to the secondary visual cortex (V2) located in front of V1. In V2, the information is recognized more three-dimensionally. In yet anterior visual areas (Figure 4), higher-level information processing concerns depth, distance, colors, movement, physical relationship, and other fine features. Researchers have found columns also in the primary somatosensory cortex responsible for the sensation of touch and the primary motor cortex responsible for movements. With the relatively universal occurence of some kind of columns, elucidating their mechanism should play an important role in understanding the structure and mechanisms of the cerebral cortex. There are still, however, many questions to be answered: Does the structure of a column vary in different functional regions of the brain? What information does each column process? How are they wired with each other? Is a column further divided into much smaller functioning units? Whether or not a column penetrates through the six layers of the cerebral cortex is one of the important questions to be answered. "Information from the thalamus goes to the fourth layer of the primary visual cortex, where the information is processed before being conveyed to the fourth layer of the secondary visual cortex. The basic principle of functioning of areas in the cerebral cortex can be simplified as to operate on information from a lower-level processing area, after which it is further conveyed to a higher-level processing area. Columns have been considered to derive from inputs to the fourth layer," said Rockland. The honeycomb structure in the first and second layers, however, does not extend to the fourth layer. In addition, the structure is as small as 0.08 mm to 0.1 mm in diameter. Our discovery has revealed that besides the columns mainly found in the fourth layer, there are other smaller column structures in the boundary between the first and second layers, apart from the fourth. This was truly an unexpected discovery, somewhat at odds with conventional assumptions. For Rockland, however, the discovery was not so unexpected because she was the person who defined in 1979 that information from V2 is conveyed to the first layer of V1: She found that in the cerebral cortex, information is not only processed from so-called lower- to higher-level areas, but also from higher- to lower-level areas. "Researchers have come to believe in recent years that the cerebral cortex compares information internally generated, such as memory, expectations, and predictions based on the person's experience and learning, with information coming in from outside. The honeycomb structure may be playing an important role in the comparison of internal and external information," stated Rockland. The first and second layers also receive information from the amygdala located in the middle of the brain. The amygdala, an evolutionarily ancient structure, controls instinctive emotions such as pleasant and unpleasant feelings. The honeycomb structure may play a role in the interaction of information and instinctive emotions. The laboratory hopes to elucidate unknown functioning of the cerebral cortex by further studying the honeycomb structure. "We are investigating which dendrites are involved in forming the honeycomb structure, which neurons the dendrites belong to, and what functions the honeycomb structure possesses," says Ichinohe. Since the honeycomb structure was discovered at the boundary of the first and second layers, this reinforces other research groups around the world who are also focusing on the functions of these layers. Rockland says, "The discovery of the honeycomb structure and the importance of the first and second layers will, I predict, lead to rewriting the textbook picture of cortical organization." |
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| Visualization of neurons | |||
 The strength of the laboratory lies in its wide anatomical experience and technique, allowing them to probe how neurons are connected to each other using various kinds of morphological methods. One of the most specialized techniques is the one to visualize an entire axon. Unlike older methods, which only stain the terminal part of an axon, these techniques allow successful staining of the entire axon from its beginning to end in detail (Figure 5).The laboratory is further developing techniques where a virus is used to inject a gene of a labeled substance (called a tracer) into a neuron. By injecting a tracer gene into the genetic region of a particular protein, for instance, they can specifically visualize neurons that produce the protein. This method will help researchers to understand its axon connectivity field. The intention of the laboratory is to further visualize neurons according to types so that they can scrutinize the circuit structure of each region to better understand the functioning principle. They are also investigating how regions of the cerebral cortex interact with each other via long distance connections to carry out their function. |
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| Investigating the structure of visual cognition | |||
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Rockland and her colleagues also are interested in how neurons are connected in the hippocampus located deep inside the brain and in the anterior-inferior temporal lobe cortex located in the temporal lobe. The hippocampus plays an important role in memory. The anterior-inferior temporal lobe cortex is the region where the final process of visual recognition is thought to occur. The Cognitive Brain Science Group, to which the Rockland laboratory belongs, is leading the world in research of these regions. Keiji Tanaka, Group Director of the Cognitive Brain Science Group, discovered columns in the anterior-inferior temporal lobe cortex as a collection of neurons that react to "moderately complicated" figures, which are not as complicated as the entire image of a concrete object. One of the Group laboratories, the Laboratory for Integrative Neural Systems (Laboratory Head, Manabu Tanifuji) is engaging in research on how these columns combine to recognize the entire image of an object, by using the optical imaging method. This method is used to detect changes in reflectance of the surface of the brain, associated with changes in level of neural activity. The Laboratory for Cortical Organization and Systematics is collaborating with the Tanifuji laboratory to elucidate how columns are connected to each other by axons in the anterior-inferior temporal lobe cortex. "It has been known that in the primary visual cortex, columns reacting to lines with similar slopes are interconnected. Based on this knowledge, we might assume that also in the anterior-inferior temporal lobe cortex, similar columns, for example those relating to a face, are bound together strongly with many axons. However, according to the research findings obtained by Hisashi Tanigawa, a research scientist at the Laboratory for Integrative Neural Systems, things are unlikely to be that simple (Figure 6). In higher level regions such as the anterior-inferior temporal lobe cortex, columns seem to be bound in a more complex way," explains Rockland. The Laboratory for Integrative Neural Systems, using the optical imaging method, is engaged in detecting activities in the third layer that sends out information. Rockland is considering that the honeycomb structure, which has its own rules of operation, may help them to understand additional aspects of how circuits are connected in the anterior-inferior temporal lobe cortex, and how this might relate to object recognition.
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| Forming new systematics | |||
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According to Rockland, in naming her team "The Laboratory for Cortical Organization and Systematics," she borrowed the word "systematics" from the following passage: "The new systematics has not emerged yet. Before it comes into existence, we need to digest, associate, and integrate new facts and ideas that have been posed to us during the last 20-30 years." This is a statement made in 1940 by J. S. Huxley, a British biologist, about the theory of evolution. Rockland considers this is also true of the current brain science. "With the recent advancement in technologies, detailed facts concerning the brain have been uncovered one after another. From these facts, I hope we can clarify principles concerning the structure and functioning of the brain and establish a new systematics. We have to be careful, however, not to be too quick to jump to a conclusion about the principles. If we rush too much, we might miss something important, and brain function is likely to hold many surprises still" says Rockland. She continues, "Researchers used to assume that any region of the cerebral cortex has basically the same structure. They therefore considered that elucidation of the principle of the structure of one region could help us understand other regions. Approximately a decade ago, however, I think they started to pay attention more to the differences among regions of the cerebral cortex." Rockland also pointed to the importance of understanding differences among different species, saying, "It is still a profound mystery whether the cerebral cortices of mice, monkeys, and humans function based on the same principle. Even a rough comparison of structures of the cerebral cortices among different species shows significant differences. Whales and dolphins, for instance, have a very thick first layer in their cerebral cortex, and little or no layer 4. As researchers are successfully decoding the genetic information of various species, I believe it is very important in the future to investigate relationships between the decoded genetic information and the structure and functioning of the cerebral cortex." Humans are the species with the most advanced cerebral cortex. The systematic research by the laboratory on how the cerebral cortex functions will surely provide us with a new perspective to consider "what human beings are." February 2005 will mark the fifth anniversary of the establishment of this laboratory within the Brain Science Institute (BSI). Rockland concluded at the end of the interview, "I think it is especially wonderful that BSI is hiring more and more young researchers who are capable of leading research projects, as well as that the Institute is placing an emphasis on basic research. I feel very honored to be able to participate in research projects at BSI, through which I can experience new adventures, and to witness their development." 
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Shedding light on the mystery of plant body plan An approach based on research using cultured cells combined with a comprehensive gene expression analysis
RIKEN Yokohama Institute |
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| Using cultured cells to understand how the body of a plant is formed | |||
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Do you know a plant called zinnia (Zinnia elegans)? This is an annual plant that belongs to the aster family. It blooms in summer with flowers in pink, yellow, and many other colors. Hiroo Fukuda, Group Director of the Morphogenesis Research Group says, "I am interested in knowing how the body of a plant is formed." The Group uses zinnia in their research to answer this question. One of the methods to analyze the mechanism of plant body formation is an approach based on mutants. In this approach, researchers first find a gene that causes mutation. They will then focus on elucidating the mechanism through a study of the functioning of the gene. The body of a plant is, however, made up of many cells that are intertwined in a complicated fashion. In addition, an abnormality in one gene exerts a variety of influences. For these reasons, it is not easy to link cause and effect. The Morphogenesis Research Group therefore uses cultured cells of zinnia. What are the advantages of using cultured cells? Fukuda explains as follows: "Using cells separated from one another makes it easier to control cellular processes uniformly. It also allows continuous observation of how they change their form and what molecules are produced by these cells. If you grasp the whole image of the process of plant body formation based on findings on a cellular basis, you can easily link causes and effects." Then, why zinnia? "The reason is very simple," says Fukuda. You can easily separate cells into single cells merely by lightly grinding the leaves of the plant in a mortar with a pestle. This is only possible with several plants including zinnia and asparagus. If you grind other plants in a mortar, their cells would be smashed. |
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| Transdifferentiation of zinnia mesophyll cells | |||
 Research using cultured zinnia cells is widely performed worldwide, where Fukuda is the pioneer. In 1980, while he was a graduate student, Fukuda discovered that photosynthetic mesophyll cells, which had been isolated from zinnia and cultured for about three days in a medium containing the plant hormone auxin and cytokinin, transformed into xylem vessel cells (Figure 1). Xylem vessel is a pathway for water and nutriment absorbed by the roots. In his experiment, a single photosynthetic mesophyll cell differentiated into a single xylem vessel cell without cell division.Conversion of a cell that has completed differentiation and acquired a certain function into another cell with a different function is called "transdifferentiation." Differentiated cells of plants demonstrate totipotency, the ability to regenerate the entire plant body. Among animals, only a limited number, including planarian, possess totipotency. Since transdifferentiation plays a major role in retaining totipotency unique to plants, the elucidation of its mechanism is essential in understanding plant body formation. When a photosynthetic mesophyll cell transdifferentiates into a xylem vessel cell, a spiral-shaped pattern of cell walls appears on its surface. While the transdifferentiation can be confirmed by observing this pattern through a microscope, what has happened in the process cannot. The process of the transdifferentiation from photosynthetic mesophyll cells to xylem vessel cells has been a mysterious black box for the last 20 years or so since its discovery. "There must be various genes expressing one after another during the transdifferentiation process from photosynthetic mesophyll cells to xylem vessel cells. I am interested in unraveling what is happening in the process on a genetic level. The number of genes possessed by higher plants is estimated to be about 20,000-40,000. My intention is to study all these genes in exhaustive detail. Due to the difficulty in performing this type of study a universities, we set up a research group four years ago when the PSC was established," says Fukuda, looking back on those days. |
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| EST library of Zinnia elegans | |||
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The Morphogenesis Research Group led by Fukuda consists of two research laboratories. The Laboratory for Gene Regulation (Laboratory Head, Taku Demura), one of the two laboratories, is playing a leading role in elucidating the mechanism of transdifferentiation using cultured zinnia cells. Arabidopsis thaliana is widely used as a model organism in plant biology. In 2000, the complete genome sequencing of Arabidopsis was completed, revealing that the plant contains approximately 26,000 genes. Meanwhile genome analysis of zinnia has not progressed. The Laboratory for Gene Regulation therefore started to construct an Expressed Sequence Tag (EST) library of zinnia. DNA in the gene region in a genome is transcribed into mRNA (messenger RNA), which is translated into protein. cDNA is mRNA that has been artificially copied into DNA. cDNA containing all the information necessary to synthesize proteins is called full-length cDNA, and a short segment of a cDNA is an EST. Why an EST library rather than a full-length cDNA library? Fukuda explains the reasons for using ESTs, saying, "Full-length cDNA is more useful in general. You can produce proteins from full-length cDNA and study their function, but you cannot with EST. EST, however, can be used as a tool to determine which gene is expressed. In addition, it takes a very much longer time and larger amounts of money to construct a full-length cDNA library than an EST library. What we need to know is which gene is activated and when in the process of transdifferentiation in zinnia, and for this a segment would be enough. We concluded speed is more important than perfection for our research." The number of ESTs contained in the zinnia EST library has now reached approximately 20,000. This, the world's largest EST library, was constructed using a technique called the "equalized library" originally developed by RIKEN. With this technique, mRNA can be effectively isolated even from a less-expressed gene. |
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| Discovery of master genes regulating transdifferentiation into xylem vessel cells | |||
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The Laboratory for Gene Regulation has developed a microarray where some 10,000 kinds of Zinnia ESTs are attached at a high density to a glass substrate. Using this microarray, the laboratory members are exploring what genes are expressed and when in the process of transdifferentiation from photosynthetic mesophyll cells to xylem vessel cells. It has been revealed so far that different gene groups are expressed at different phases of transdifferentiation (Figure 2). "We thought there must be a gene that plays a key role in regulating the expression of every gene group. Based on this assumption, we searched for a "master gene" playing such a role," says Fukuda. "We have recently found a master gene that regulates the transdifferentiation into xylem vessel cells. With this gene inserted, cells of different kinds transformed into xylem vessel cells (Figure 3)." First, we found a candidate of the master gene in zinnia. Immediately after detecting an interesting gene in zinnia, researchers on the laboratory start searching for genes with similar base sequence in other species, an activity that has become possible thanks to genomic databases available for different species. In this way, the researchers found in Arabidopsis more than one gene that possess a base sequence similar to the one in the gene found in zinnia. When the researchers inserted one of the genes into an Arabidopsis plant, cells of different kinds in the plants transdifferentiated into xylem vessel cells, indicating that master gene may play a role in regulating the transdifferentiation into xylem vessel cells regardless of differences in species. After more than two decades, light has finally been shed into the mysterious black box. Fukuda says, "The new discovery was only possible by a successful combination of our research using cultured cells over the past years and comprehensive genetic analysis unique to RIKEN." The laboratory members are to analyze in more detail the functioning of the master gene and to search for other master genes. Xylem vessels are pathways of water and nutriment absorbed by the roots. Nutriments produced by photosynthesis flow through sieve tubes. Between xylem consisting of vessels and phloem consisting of sieve tubes is cambium. The xylem, phloem, and cambium collectively form a vascular bundle, an important structure that transports information and materials throughout the body. The bodies of trees and plants are mainly composed of these vascular bundles. Fukuda considers it important to understand the entire vascular bundle, including vessels, in elucidating how the body of a plant is formed. He says, "We have started our research with the transdifferentiation into xylem vessel cells. Based on the current research, our goal is to elucidate the mechanism of how the vascular bundles are formed on a genetic level."
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| Application to biomass | |||
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Fukuda points out, "Oil will eventually be depleted. Without using biomass, humans will not be able to survive the 21st century. One of the most important issues for human beings is therefore how to make the best use of biomass. I am confident that our research will be of most use in solving this problem." Biomass is an organic resource derived from living organisms such as foodstuffs, materials, and fuel. The biomass on the surface of the Earth mainly consists of trees, the most part of which is made up of the xylem. In this context, it is expected that elucidating a transdifferentiation mechanism that leads to transformation into xylem vessels and other cells in the xylem will enable us to promote the growth, increase the volume, and change the properties of the xylem. With the application of their research outcomes to biomass as their future goal, the laboratory has also started a study using poplar, a tree plant widely used in laboratory studies. The entire genome sequencing of poplar is expected to be completed around the beginning of the year 2005, following Arabidopsis thaliana and Oryza sativa. In their research, a poplar gene similar to the master gene discovered in Zinnia is used to see whether it actually promotes the growth of xylem vessel cells. Fukuda has a definite feeling that its practical application will become feasible within the foreseeable future. |
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| Using cultured BY-2, a tobacco cell line, as a plant for producing substances | |||
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The Laboratory for Structural Construction (Laboratory Head, Ken Matsuoka), another laboratory of the Morphogenesis Research Group, is engaging in research using cultured tobacco cell line BY-2. The BY-2 cell line, developed in 1960 by the former Japan Tobacco Monopoly Corporation (the current Japan Tobacco Inc.), proliferates at a high speed, with a 60-100-fold increase in a week. This is currently one of the most frequently-used plant cultured cells around the world. The laboratory is constructing an EST library containing approximately 20,000 genes obtained from the BY-2 cell line, based on the technique acquired from their experience through research on zinnia. They are also developing a microarray using the ESTs to analyze what genes are expressed and when in the process of BY-2 proliferation. Disclosure of exhaustive and comprehensive information about the genes and their expression will help enhance the value of BY-2 as a research tool, subsequently raising the level of the research. "Our laboratory is leading the world in research using zinnia cultured cells and tobacco BY-2 cells," Fukuda says confidently. The laboratory is especially focusing on intracellular transport systems of materials in studying plant body formation. Material building plant cell walls are mainly produced in the Golgi apparatus and endoplasmic reticulum and transported to cell walls. How are they transported before forming a cell wall? What kind of enzymes work in the process? Nobody has yet answered these questions. Fukuda and his colleagues are seeking to elucidate gene-level mechanisms of cell wall formation using cultured tobacco cell line BY-2. Fukuda has a plan to expand the use of BY-2 in the future. "I am considering using cultured BY-2 as a manufacturing plant for substances. The cell line grows rapidly. In addition, it is easy to insert a gene into BY-2. If you insert genes encoding synthetic enzymes for a substance you want to produce and its substrate, you can mass-produce the substance within BY-2." |
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| Research that leads us to an exit | |||
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"I'm interested in finding the basic mechanisms of an organism. I want to know more about how a cell lives and how a plant lives, and to find out the mechanisms of newly-discovered aspects of their life." Fukuda does have his own strategy. He says, "Our advantage is that we took a unique approach by starting our research with cultured cells of zinnia and BY-2, not with Arabidopsis, which is commonly used as a model plant. We have gathered information on these plants on a cellular basis. We can always refer to research findings on Arabidopsis, if necessary. By cross-referencing information about zinnia and BY-2 with information about Arabidopsis, just like playing catch, you will be able to obtain more new information." Fukuda concluded saying, "People often say that botanical science does not have an exit, meaning that it does not lead us to practical applications. Unfortunately, I think it is true. While basic science is important, its value will not be acknowledged unless you combine it with research that leads to practical applications. We need to be strongly aware of the necessity of practical application in future (Figure 4), and I believe this to be one of the important roles of PSC.
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