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Planned collaborative project

Planned collaborative project themes are selected by NIPS, which are based on requests from researchers. Until 2007, there were two themes, “Physiological and neuroscientific studies into genetically modified model animals” and “Biomolecular sensors and physiological function.” Additional themes were added in 2008, with “Functional and morphological analyses of cells and tissues by multi-photon microscopy” and “Medical and biological applications of phase-contrast cryo-electron microscopy” (name changed to “Medical and biological applications of cutting-edge electron microscopy” in 2011), and in 2009 with, “Behavioral analysis of mouse and rat”. Also, “Analysis of metabolic physiology for mouse and rat” began in 2011, while “Transfection study with primates,” “Analysis of fluctuations in function research in life science,” and “Multidisciplinary study of neural information” began in 2012. Also, “Transfection study with viral vector neurological system” was started in 2013. Furthermore, “Purification of supra-molecular complexes and analyses of their constituents by mass spectrometry” was started in 2016, and “Analyses of dynamic aspects of the function and structure of membrane proteins” in 2017. All these themes cover the most talked about scientific topics today, and are areas where NIPS is considered to be a frontrunner in Japan. We expect to receive many new proposals.
Two projects, ”Analysis of fluctuations in function research in life science” and “Multidisciplinary study of neural information” were closed in 2015, due to the finish of the related NINS projects.
“Behavioral analysis of mouse and rat” was closed due to the shutdown of the Section for Behavior Patterns. In 2016, NIPS performed only the collaborative experiments carried over from last year.

In regards to the proposal agenda, long discussions had been carried out at both faculty meetings and work meetings in 2012. The agreed requirements are as follows.
1) Proposals should clearly state the aim and experimental design of the research project and should be completed within five years. However, depending on the state of the research, an extension period may be granted after the initial five years.
2) Proposals should specifically state the research area of interest. Broad themes will not be accepted.
3) There will be a limit to the number of proposals accepted. Each general collaborative research area category and research facility will accept five projects each at most, in principle.

The details of the planned collaborative research are as follows.

“Physiological and neuroscientific analysis of genetically modified model animals.”

Since genetically modified model animals are extremely effective for gene function analysis at individual level, they are widely used in the field of life sciences. The recent engineering required to create such model animals has taken huge leaps forward; e.g., a new genome-editing tool (CRISPR/Cas9 system) can relatively easily cut arbitrary sequences on the genome. Section of Mammalian Transgenesis at the Center for Genetic Analysis of Behavior in NIPS has established the latest technology such as the CRISPR/Cas9 system capable of providing endogenous genetic modification to mice and rats. Our staffs familiar to not only physiology and brain science but also reproductive biotechnology, have greatly contributed to researchers all across the country by providing technology to create genetically modified model animals. We can support cooperative studies by providing the technologies to develop adoptive models such as transgenic or knock-out mice and rats. In the calendar year of 2018, we have created a total of 19 transgenic or knock-out mouse and rat lines under 9 collaborative research projects. We will continue to work on the requested creation of genetically modified model animals by applying the new genome-editing tools.

“Analysis of metabolic physiology for mice and rats.”

The Section of Metabolic Physiology was set up in 2010, and the planned collaborative research project, “Metabolic physiology analysis of mice and rats,” had started in 2011. Since then, researchers from within and outside NIPS have been looking at the following topics concerning genetically modified animals.
1) Measuring neural activity of individual neurons associated with motor function while awake.
2) Circuit behavior imaging of flavin and hemoglobin intrinsic signals in the brain using voltage-sensitive dyes.
3) Measuring food intake and energy consumption during free movement.
4) Measuring body temperature, pulse rate, and blood pressure
5) Measuring the cardiac function and blood flow volume of mice in vivo or in vitro.  
Five collaborative research projects with researchers outside NIPS were conducted in 2018, and seven projects are now scheduled in 2019.

”Ultrastructure analysis of biological specimens by cutting-edge electron microscopy.”

One phase-contrast electron microscope (PC-TEM) and two serial block-face scanning electron microscopes (SBF-SEMs) are mainly used for this joint research program. PC-TEM developed by NIPS shows the best performance when combined with a rapid-freezing sample preparation method. Under this condition, it is possible to study three-dimensional structures of unstained biological specimens, including isolated proteins, viruses, bacteria, cultured cells, and tissues, to more or less their true state with 1 nm spatial resolution. On the other hand, SBF-SEMs are used for the studies of ultrastructural analysis of thick biological specimens, like a brain tissue. The specimens embedded in the plastic resin are sliced by a diamond-knife and imaged by SEM continuously. Finally, the three-dimensional ultrastructure of the specimens is rebuilt at dozens of nanometer resolution. The program support studies by using these state of the art electron microscopes. In 2018, 18 projects were carried out, and 14 are now scheduled in2019.

“Functional and morphological analyses of cells and tissues by multi-photon excitation microscopy.”

Two-photon excitation fluorescence microscope is a less invasive method for studying the microscopic structure and functions of cells in deep tissues of biological organisms. Currently, our institute has three upright two-photon excitation microscopes, and these allow us to observe the structure in depth of one millimeter with a spatial resolution of a micrometer. Since the maintenance of two-photon microscope is complicated, NIPS is the only institute which can provide the opportunity for collaborative research with the high-quality experience. Furthermore, we recently build the two-photon fluorescence lifetime microscope system which enables us to observe the intermolecular interactions and the activity of signaling protein in a living cell in the deep tissue. We are also working on single-molecule imaging using quantum dot in a combination of a fluorescence microscope. Using these "cutting-edge methods," we have conducted the collaborative researches. Recent successes are particularly in vivo Ca2+ imaging, and long-term imaging of neurons in living mice.
In 2018, four planned collaborative projects were carried out, and four were scheduled in 2019. We also discussed collaborative research with over ten groups and introduced our multi-photon excitation microscopes to over ten groups.

“Development and supply of viral vectors and gene-transfer to primates.”

Advances in technology to control molecular functions or change neural activity by inserting certain genes into primate brains using virus vectors can lead to major possibilities. Getting to do such research, however, requires a long list of equipment and facilities to enable researchers to develop do things such as develop vectors, or insert vectors. A planned collaborative research project “Transfection study with primates” was launched in 2012 so that researchers could share their resources, and work together to unravel mysteries about higher brain functions and pathological conditions. In 2013, five projects were carried out, and five projects were carried out in 2014.
The key point of the experiments is the development of suitable viral vectors. Also, viral vectors are useful, not only for primates but also for other animals. Thus, a planned collaborative project “Gene transfer into the nervous system using viral vectors” was started in 2013. In Section of Viral Vector Development, we promote the collaboration with many laboratories by providing various serotypes of AAV vectors, conventional lentiviral vectors, and highly efficient retrograde gene transfer vectors. Moreover, we proceed with the collaboration to exploit the more advantageous viral vectors. Up to 2014, we provided more than 100 viral vectors for other laboratories and performed two planned collaborative research in 2013, and 4 in 2014. At present, very intriguing research results are being obtained. In 2015, the two projects were merged as “Development and supply of viral vectors and gene-transfer to primates. ” The three examples of the achievements are as follows. 1) Virus vectors helped to identify system circuits that compensated motor functions after spinal cord injury in macaque monkeys. 2) Virus vectors revealed the property of subnetwork composed of excitatory and inhibitory neurons in layer 5 of the rat frontal cortex. 3) Virus vectors identified a specific subset of neurons commanding the dietary preference for carbohydrate over fat in mice.
In 2018, 4 research were performed, and 16 are now scheduled in 2019.

“Purification of supra molecular complexes and analyses of their constituents by mass spectrometry.”

To understand the function of proteins in vivo, it is necessary to identify the constituents of supra molecular complexes precisely.  Therefore, there are gradually increasing needs for the support to perform purification of protein complexes from tissues and cells, and to identify constituents of the complex and the target antigens in auto- immune diseases by mass spectrometry. This project was newly started in 2016 to respond to the needs. In2018, one research were performed, and one is now scheduled in 2019.

”Analyses of dynamic aspects of the function and structure of membrane proteins”

Functional membrane proteins such as ion channels and receptors are strictly designed molecules. They, at the same time, show dynamic changes of the structure and function depending on the situation. To analyze the dynamics aspects by electro-physiological and opto-physiological experiments using in vitro expression systems, we perform this planned collaborative project. In 2018, we conducted 6 research projects, and 6 are now scheduled in 2019.

Large facilities and equipments

The National Institute for Physiological Sciences(NIPS)...