13. JOINT RESEARCHES

Outline

National Institute for Physiological Sciences (NIPS), one of Inter-university Research Institutes, is conducting various types of cooperative studies such as (1) General collaborative project (2) Planned collaborative project focusing on several important themes at each period, and (3) Cooperative studies using large facilities. As shown in the following tables, many cooperative studies have been done every year. In fact, 60 cooperative studies and 35 cooperative studies using large facilities in total are now being conducting this year, and excellent and fruitful results have been already gotten.

Another important main work of cooperative studies of NIIPS is NIPS research meeting. Twenty six NIPS research meetings will be held this year. The number of NIPS research meeting is much larger than other two institutes at Okazaki. Unlike usual congresses or meetings, oral presentations are mainly done with a long time of Q & A. In addition, since the number of participants is relatively small, more detailed and focusing discussion can be done. Since several official big projects supported by Ministry of Education, Science, Sports, Culture and Technology of some official Academic Associations were founded based on the NIPS research meeting, NIPS research meeting is considered to be very important and useful by researchers community. NIPS International Workshop is beginning from last year. Inviting several guests from foreign countries, all talks are presented in English. This new style of workshop should be promising.

1. General collaborative project

General collaborative project and Planned collaborative project are the cooperative studies by researchers in other universities or Institutes with researchers at NIPS. Approximately 30 to 40 proposals were accepted previously, but with an increase of activities of cooperative studies, 60 and 65 proposals were accepted last year and this year, respectively.

2. Planned collaborative project

Planned collaborative projects are determined by NIPS considering requirements by researchers. "Physiological and neuroscientific research of gene-modified animal models" and "Biomolecular sensors and their physiological functions" have been established as first planned collaborative projects. "Medical and biological applications of phase-contrast cryoelectron microscopy" and "Functional and morphological analyses of cells and tissues by multi-photon microscopy" began 2008, and "Systematic analysis of behaviors of mice and rats" was added from 2009. These three themes are one of the hottest topics of sciences, and are that research activities at NIPS is considered to be very high and excellent. Many excellent new proposals are expected.

Two new Planned collaborative projects are as follows.

"Functional and morphological analyses of cells and tissues by multi-photon microscopy" provides the real images of various fine structures located in the deeper areas of the tissues and living animals with less damage.

Thus, this newly advanced imaging technique is rapidly attracted in the life science field and expected to be a breakthrough for innovative research findings especially in Europe, USA and Japan.

Although few laboratories are practically applying this tool because of the technical difficulties in its maintenance and adjustment, our institute (NIPS) is steadily running, and uniquely offering exciting collaborations by using this cutting-edge apparatus with academic and industrial laboratories in Japan.

Currently, we run two upright and two inverted two-photon microscopy, which provide the highest class imaging quality all over the world.

In addition, we have improved the laser light path as a joint project with an optics company under the support from Japan Technology and Science Agency. This achievement allowed it possible to observe fine structures in the areas up to 1 millimeter depth from the surface with resolution of less than 1 micrometer.

We also succeeded in in vivo Ca²⁺ imaging obtained from various neurons and glia, and in establishing a special equipment attached onto the skull to help long-term imaging of same fine structures over several weeks in the same animals.

"Medical and Biological Applications of Phase-Contrast Cryo-electron Microscopy"

A novel electron microscope recently developed in NIPS is world-wide unique as the one and only running phase-contrast electron microscope to date. Its imaging capability, particularly enhanced when combined with quick freezing sample preparation, enables us to shoot photos in a spatial resolution of 1nm for unstained structure-preserved biological specimens. Encouraged by past experiences of collaborative works managed inside the Nano-Structure Physiology Division, this planned joint study has started from 2007 and made the novel facility open to biological and medical societies. It is also powered up by a new capability of phase contrast electron tomography from 2009. Challenging researches are to be awaited in such applications as structural studies for receptors and channels, high resolution molecular processes in whole bacterial cells and nanometer morphology for mamallian cells and tissues.

Planned collaborative project on "Behavioral Analysis of Mouse and Rat" has been started from 2009.

It has become possible to correlate the gene expression with the behavior by using gene modified animals. However, to accomplish this, multiple behavioral tasks should be conducted in a reproducible manner. It is difficult to conduct these experiments independently by the individuals. The National Institute for Physiological Sciences has thus founded the Section of Behavior Patterns in the Center for Genetic Analysis of Behavior and invited Professor Miyakawa to become the first adjunct professor of the section. This section will be responsible for the Planned collaborative project on "Behavioral Analysis of Mouse and Rat". In year 2009, only the analysis on mice will be available.

3. NIPS research meeting

Twenty five NIPS research meetings will be held and more than 1,000 participants will participate in them this year. Unlike usual congresses or meetings, oral presentations are mainly done with a long time of Q & A, focusing on concrete themes. In addition, since the number of participants is relatively small, more detailed and focusing discussion can be done. Since several official big projects supported by Ministry of Education, Science, Sports, Culture and Technology of some official Academic Associations were founded based on the NIPS research meeting. NIPS research meeting is considered to be very important and useful by researchers community.

4. NIPS International Workshop

To promote NIPS research meeting more internationalized, NIPS International Workshop has been settled from 2008. In this workshop, several advanced scientists are invited from abroad and make an intense discussion with Japanese speakers and participant on the selected topics. All presentations and discussions will be held in English. In 2008, the workshop entitled by "From photon to mind: Advanced Nonlinear Imaging and Fluorescence-based Biosensors" was accepted and held at Okazaki Conference center. In this two-day workshop, 4 foreign and 9 Japanese speakers presented their recent achievements. In addition, 12 poster presentations were held by young researcher. More than 130 participantes made an intense discussion on the topics of recent advanced laser apparatus, imagings of biomolecules, various structures in the brain and their functions, as well as newly developed fluorescent molecules.

5. Co-operative study by high voltage electron microscopy

The high voltage electron microscope (H-1250M) specially designed for the exclusive use for medical and biological field in Japan is available since 1982.

This is only a high voltage electron microscope in the biological field in Japan, the collaborative research programs are running from universities etc. on three projects: 1) three-dimensional analysis of fine structures of biological specimens till about 5mm thick 2) high resolution observation of biological specimens 3) observation of biological specimens in their natural conditions.

Average availability of the H-1250M is about 80% since installation. Almost half of the total number of days used since 1982 was utilized by researchers from other universities and institutes including from other countries. In these respects, the H-1250M has worked as a center of high voltage electron microscope in the medical and biological field in Japan.

In 2008, 13 projects were accepted and 66 days were used by researchers from outside of the institute, 65 days by researchers inside.

6. Co-operative study by functional magnetic resonance imaging

Collaborative studies using magnetic resonance imaging (MRI) have been undertaken for noninvasive three-dimensional structural and functional (hemodynamic and metabolic) changes of living organism, including brain activation studies, so called functional MRI. NIPS installed new 3 Tesla machine in 2000. The 3 Tesla machine is twice as sensitive as 1.5 Tesla standard one regarding the blood oxygen level dependent contrast, advantageous for functional MRI. The machine is equipped with the device that allows non-human primate studies both for anatomical and functional investigations. Particularly for functional MRI, whole procedures from experimental design, data collection, to statistical imaging analysis are systematically tailored to provide high quality service to the collaborators inthe neuro-physiology / neuroscience community.

7. Co-operative study by magnetoencephalography

Since 1991 when the new MEG multi-channel MEG (magnetoencephalography) machine was installed at NIPS, NIPS have done good works using MEG and reported many excellent papers as the pioneer of MEG studies in Japan. In addition, many researchers in universities or institutes where MEG machine was not installed yet visited NIPS and did good cooperative studies. NIPS was and is still now only one institute which provides an opportunity to use MEG machine to researchers outside as cooperative studies using large facilities. NIPS installed new whole-head type MEG machine in 2002. This is used just for research objective, though MEG machines in other universities are used for both clinical and research works, mainly the former. At present, basic MEG studies at NIPS is evaluated as one of the best not only in Japan but also in the world. Cooperative studies using MEG are classified into two themes: (1) Investigation of higher brain functions such as judgment, memory and learning, and (2) Investigation of sensory and motor functions. One of the most important issues for MEG study at NIPS is corroborative studies using other neuroimaging methods such as functional magnetic resonance imaging (fMRI), transcranial magnetic stimulation (TMS) and near infra-red spectroscopy (NIRS).

RECENT ADVANCE IN MULTIPHOTON EXCITATION MICROSCOPY

Outline : in vivo imaging of neurons and glia

Multiphoton excitation of photo-sensitive molecules, e.g. fluorescent molecules, at a limited area is introduced by using a high power femtosecond pulse laser. The advantage of multiphoton excitation is to employ the light with longer wave length that reaches deeper into the tissue with less damage. In addition, multiphton excitation could be occurred at a limited area having a high density of photon under the objective lens. Thus, this method allows us to observe various fine structures at deep tissues in an in vivo and in vitro preparation. We have improved the light-path and objective lens, and achieved the clear images of fine structures, e.g. dendrites, axon, postsynaptic spines and presynaptic boutons of the mouse brain as deep as 1mm from the surface. In addition, we also succeeded in repeatedly observed the same fine structures over a month in living mice.

Using this recent advanced technique on fluorescent labeled neurons and microglia we demonstrate that the resting microglial processes make brief (~5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent. Following transient cerebral ischemia, the duration of these microglia-synapse contacts are markedly prolonged (~1 hour). Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed towards synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear following prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections in the damaged circiuts. Remodeling neuronal circuits by microglia may lead to novel therapies for treating brain injury that target microglia. In addition, we established the approach to make in vivo image of the mobility of peripheral immune cells, and of blood flow in the various vessels in size.