Graduate School of Medicine, Kyoto University
Outline of Research Activities
The primary focus of research has been on the neuronal circuitry, synaptic organization, and development and plasticity of neuronal connection in the central nervous system of mammals, including man. Various pathways in the brain and spinal cord are analysed with light and electron microscopes by means of tracer techniques, immunocytochemistry, in situ hybridization histochemistry, and intracellular recording/staining method. Recently, the efforts are strongly focused on the morphological analysis of local circuit or microcircuit in the cerebral cortex, basal ganglia and thalamus, using genetically modified animals as well as the conventional methods listed above.
1. Local circuitry in the CNS
Local neural circuitry is considered to be the basis of the higher functions executed by the CNS. The laocal connections between neurons were first examined by the Golgi impregnation technique which enabled us to completely visualize the input site of neurons,i.e. dendrites. However, this method has some techinical limitations such as nonselective sampling of neurons and poor labeling of axons. From then on many other techniques including electron microscopy and intracellular staining have been introduced to analyze the local circuitry of the CNS. However, electron-microscopic studies are unsuitable for surveying neuronal circuitry within a CNS region because the magnification factor is too large to observe the whole structure of the region. Instracellular starting of two neurons, though its excellent visualization of dendrites and axons, suffer from the same sampling problem as the Golgi impregnation method does. If a new technique for Golgi-like visualization of dendrites and axons of functionally grouped neurons is developed, the technique, combined with the conventional methods such as instracellular staining, will be very helpful in analyzing local neuron-to-neuron connections within a CNS region.
2. Tools for the morphological study of the CNS
Recently, we have developed a new retrograde axonal tracing method with tetramethylrhodaminedextran amine, which labeled soma and dendrites of cortical projection neurons in a Golgi-stain-like manner (publication #67). Combining this techique with intracellular staining, we have studied the local circuit of the cerebral cortex, and found that the information of layer III is transferred more efficiently to layer V corticospinal projection neurons than to layer VI corticothalamic neurons in the motor cortex (101). Furthermore, we are now trying to develop molecular-biological tools for analysis of local neuronal circuit. It was successful, using viral vectors, that a plasma membrane-targeted protein was expressed in vivo to visualize neurons in a Golgi-stain-like fashion with an antibody to the protein (107). Graduate students are producing the genetically altered mice that express similar membrane-targeted proteins in a specific subset of neurons by use of specific promoter regions of the gene. When they succeed in the production, they will analyse the local neural circuitry by applying the intracellular staining method to the transgenic or knock-in mice, or by labeling two different subsets of neurons in a Golgi-stain-like manner with F1 offspring of two lines of the mice that produce different membranetargeted proteins, respectively.
3. Chemical characteristics and neural circuit in the CNS
Hodological and chemical characteristics of neuronal circuit has long been studied from the time of the former chair, Prof. Mizuno. Mr. Furuta and Dr. Fujiyama are now studying chemical and hodological characteristics of basal ganglia neurons (106). Prof. Kaneko and Dr. Fujiyama have started the immunocytochemical and in situ hybridization studies of vesicular glutamate transporters, which is the best markers for glutamatergic neurons. Guest investigators from China, together with Prof. Mizuno and us, continue collaborative works on the trigeminal nervous system.
4, Development of the CNS
Dr. Tamamaki is investigating the mechanism of the neuronal development in the cerebral cortex, using molecular-biological techniques such as viral vectors and transgenic mice. He has succeeded in the production of viral vectors which visualize infected neurons in a Golgi-stain-like manner (107). Using the viral vector, he found the cell migration from the corticostriatal angle to the basal telencephalon through lateral cortical stream in rat embryos (112). He is now studying neural stem cells and their development in the mouse embryo with the viral vectors, and obtaining the evidence that radial glias are actually neural stem cells.
The research in system neuroanatomy was largely developed in the 1970s and 1980s by the introduction of new retrograde and anterograde tracers, immunocytochemistry and in situ hybridization histochemistry. We now have a large body of information on the hodological and chemical charateristics of CNS neurons except for the knowledge on the local circuit. Thus, the question of local neuronal circuitry is a sort of "final frontier" in the field of system neuroanatomy, although it is a very tough question and it needs a long time to be solved. After elucidation of local neuronal circuitry, we believe that the time will come when the behavior of a neural circuit will be analyzed with a simulation model based on the real local neural networks. We will there compare the predictions from the simulation model with the results obtained by electrophysiological studies, like in the field of theoretical and experimental physics. Actually, our department is now collaborating with researchers in the field of physics, engineering and informatics, and developing a simulation model of neurons and their networks. The functional principales of the CNS, especially those of the cerebral cortex, will only be discerned in the future through this kind of efforts of system neuroscientists.
Graduate school of Medicine,