Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution Waseda University
 
2.Research Area Life Sciences
 
3.Research Field Cellular Signaling
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96L00310
 
6.Title of Project Signaling Mechanism in Neuronal System

7.Projetct Leader
Name Institution,Department Title of Position
Yoshioka, Tohru Waseda University, School of Human Sciences Professor

8.Core Members

Names Institution,Department Title of Position
Shibata, Shigenobu Waseda University, School of Human Sciences Professor

9.Cooperating Researchers

Names Institution,Department Title of Position
Ikeda, Masayuki Waseda University, Advanced Research Institute for Science and Engineering Lecturer
Hirono, Moritoshi Waseda University, Advanced Research Institute for Science and Engineering Assistant
Moriya, Tkahiro Waseda University, Advanced Research Center for Human Sciences Assistant

10.Summary of Research Results

During the research period we have focused our interest into visualization of signaling pathway using several kinds of knock out mice, such as NR2A(-/-), NR2C(-/-), NR2AC(-/-), PLCβ4(-/-) and GFAP(-/-). Neuron of the mice we observed here are in the hippocampus, thalamus, cerebellum and amygdala, and we examined the kinetics of neuron by patch clump experiment, Ca imaging, in-site hybridizations, and in-gel-assay. Abnormal behaviors of the knock out mice were assayed by eye-blinks reflex conditioning, fear conditioning, circadian rhythm and sleep (slow wave sleep, rem sleep and awake). Protein molecules we managed in the experiment are PLCβ4 and voltage sensitive Ca channels. Therefore the research project we carried out in this period can be expressed briefly as Ca signaling in the neuron.
We have found that PLCβ4 in the rostral part of the cerebellum are essential to form LTD and eye blink reflex conditioning and it is also very significant that PLCβ4 is localized at thalamus to make REM sleep. On the other hand PLCβ4 conditioned in supra chiasmatic nucleus (SCN) is not useful to regulate circadian rhythm.
For the future development of the neuroscience we have established two kinds of novel technologies, which are thermal imaging and transfection of Ca sensitive fluorescent protein in the organella, such as ER, SR and Mitochondria. Thermal imaging reflects which proteins in the molecular circuit are involved in the desired signal transduction. Cameleon and Pericam, both of which are initially developed by Dr. Miyawaki, were transfected in the neuron as well as in the neuronal cell lines and successfully observed Ca correlation between cytosol and other kinds of organella. Using these newly developed techniques, every area of the neuroscience will be promoted largely in the near future.

11.Key Words

(1)knock out mice、(2)Ca signaling、(3)Voltage dependent Ca channel
(4)Ca activated K channel、(5)Phospholipase C、(6)Associative learning
(7)Eye blinks reflex、(8)Circadian Rhythm、(9)Sleep

12.References

[Reference Articles]
Author Title of Article
Ikeda, M. Cyclic AMP regulates the calcium transients released from IP3-sensitive stores by activation of rat κ-opioid receptor expressed in CHO cells
Journal Volume Year Pages Concerned
Cell Calcium 29(1) 2001 39-48

Author Title of Article
Moriya, T. N-Methyle-D-Asparate Receptor subtype 2C Is Not Involved in Circadian Oscillation or Photoic Entrainment of the Biological Clock in Mice
Journal Volume Year Pages Concerned
J. Neurosci. Res. 61 2000 663-673

Author Title of Article
Sugiyama T. A novel function of synapsin II in neurotransmitter release
Journal Volume Year Pages Concerned
Molecular Brain Research 85 2000 133-143

Author Title of Article
Moriya T. Close linkage between Calcium/Calmodulin Kinase II α/β and NMDA-2A receptors in the lateral amygdala and significance for retrieval of auditory fear conditioning
Journal Volume Year Pages Concerned
Eur. J. Neurosci. 12 2000 3307-3314

Author Title of Article
Hirono, M. Sapecin B alters properties of rapidly inactivating K+ channels in rat pituitary GH3 cells
Journal Volume Year Pages Concerned
Cell Physiol. Buochem. 10 2000 177-186

Author Title of Article
Tojima T. Three-dimensional Characterization of Interior Structures of Exocytotic Apertures of Nerve Cells Using Atomic Force Microscopy
Journal Volume Year Pages Concerned
Neurosci. 101(2) 2000 471-478

Author Title of Article
Jiang Z. Pre-and postsyanptic actions of serotonin on rat suprachiasmatic nucleus neurons
Journal Volume Year Pages Concerned
Brain Res. 866 2000 247-256

Author Title of Article
Ikeda, M. PLC β4 - independent Ca2+ rise via muscarinic receptors in the mouse suprachiasmatic nucleus
Journal Volume Year Pages Concerned
Neuro Report 11 2000 907-912

Author Title of Article
Moriya, T. Involvement of glial fibrillary acidic protein (GFAP) expressed in astrogial cells in circadian rhythm ander constant lighting conditions in mice
Journal Volume Year Pages Concerned
J Neurosci Res 60 2000 212-218

Author Title of Article
Kouzu, Y. Mutant Mice Lacking Ryanodine Receptor Type3 Exhibit Deficits of Contextual Fear Conditioning and Activation of Calcium/ Calmodulin-Dependent Protein Kinase II in the Hippocampus
Journal Volume Year Pages Concerned
Mol Brain Res. 76 2000 142-150

Author Title of Article
Megumi, T. Developmental Change of GABAA Receptor-Mediated Current in Rat Hippocampus
Journal Volume Year Pages Concerned
Neuroscience 96 2000 507-514

Author Title of Article
Shibata, S. Melatonin Normalizes the Re-entrainment of Senescence Accelerated Mice (SAM) to a New Light-dark Cycle
Journal Volume Year Pages Concerned
Advances in Experimental Medicine and Biology 460 1999 261-270

Author Title of Article
Sugiyama, T. Localization of Phospholipase C β Isozymes in the Mouse Cerebellum
Journal Volume Year Pages Concerned
Biochem. Biophys. Res. Commun. 265 1999 473-478

Author Title of Article
Yoshioka, T. Imaging analysis of neuronal signaling
Journal Volume Year Pages Concerned
Curr. Topics Neurochem. 2 1999 105-121

Author Title of Article
Kobayashi, T. Ethanol Opens G-Protein-Activated Inwardly Rectifying K Channels
Journal Volume Year Pages Concerned
Nature Neurosci. 2 1999 1091-1097

Author Title of Article
Tsuchiya, K. Postictal Blickade of Ischemic Hippocampal Neuronal Death in Primates Using Selective Cathepsin Inhibitors
Journal Volume Year Pages Concerned
Exp. Neurol. 155 1999 187-194

Author Title of Article
Allen, C. N. Orphanin-FQ (OFQ) Modulates the Activity of Suprachiasmatic Nucleus Neurons
Journal Volume Year Pages Concerned
J. Neurosci. 19 1999 2152-2160

Author Title of Article
Hirono, M. Role of Ca2+-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells
Journal Volume Year Pages Concerned
Cell Calcium 25 1999 125-135

Author Title of Article
Kojima H. Improved Infrared DIC-Video Microscopy and Its Application to Unstained Living Preparations
Journal Volume Year Pages Concerned
Bioimages 6(1) 1998 9-19

Author Title of Article
Hirono, M. Phosholipase C-Independent Group I Metabotropic Glutamate Receptor-Mediated Inward Current in Mouse Purkinje Cells
Journal Volume Year Pages Concerned
Biochem. Biophys. Res. Commun. 251 1998 753-758

Author Title of Article
Sakakibara, M. Evidence for the involvement of inositol trisphosphate but not cyclic nucleotides in visual transduction in Hermissenda eye
Journal Volume Year Pages Concerned
J. Biol. Chem. 273 1998 20795-20801

Author Title of Article
Yamashima, T. Inhibition of ischaemic hippocampal neuronal death in primates with cathepsein B inhibitor CA-074:a novel strategy for neuro protection based on calpain-cathepsin
Journal Volume Year Pages Concerned
Eur. J. Neurosci. 10 1998 1723-1733

Author Title of Article
Zohar, O. Thermal Imaging of Receptor-Activated Heat Production in Single Cells
Journal Volume Year Pages Concerned
Biophysical J. 74 1998 82-89

Author Title of Article
Kishimoto, Y. Conditioned eyeblink response is impaired in mutant mice lacking NMDA reccepter subunit NR2A
Journal Volume Year Pages Concerned
NeuroReport 8 1997 3717-3721

Author Title of Article
Suzuki N. Facilitation of Ca2+ action potencial frequency by a small G protein Rab3A in rat pituitary GH3 cells.
Journal Volume Year Pages Concerned
B. B. R. C. 235 1997 331-335

Author Title of Article
J de Barry Time-resolved imaging of prptein kinase C activation during sea urshhin egg fertilization
Journal Volume Year Pages Concerned
Exp. Cell Res. 234 1997 115-124

Author Title of Article
Suzuki, N. Sapecine B, a Novel Fly Toxin, Blocks Macroscopic K+ Currents in the GH3 Rat Pituitary Cell Line
Journal Volume Year Pages Concerned
J. Am. Physiol. 273 1997 c289-c296

Author Title of Article
Inoue, H. Purification of a Regulatory Subunit of Type II cAMP-Dependent Protein Kinase from Drosophia Heads
Journal Volume Year Pages Concerned
Biochem. Biophys. Res. Commun. 235 1997 223-226

Author Title of Article
Olds, J. L. Imaging of protein kinase C activation in living sea urchin eggs after fertilization
Journal Volume Year Pages Concerned
Dev. Biol. 172 1996 675-682

Author Title of Article
Inoue, H. A novel function for nucleoside diphosphate kinase in Drosophila
Journal Volume Year Pages Concerned
Biochem. Biophys. Res. Commun. 218 1996 887-892

Author Title of Article
Kasai, H. Two components of exocytosis and endocytosis in PC12 cells studied using caged-Ca2+ compounds
Journal Volume Year Pages Concerned
J. Physiol. 494(1) 1996 53-65

Author Title of Article
Yamashima, T. Transient Brain ischemia provokes Ca2+, PIP2, and calpain responses prior to delaved neuronal death in monkeys
Journal Volume Year Pages Concerned
Eur. J. Neurosci. 8 1996 101-113

[Reference Books]
Author Title of Book
Yoshioka, T. Introductory Review: Synaptic Plasticity and Modulation. in Slow Synaptic Responses and Modulation
Publisher Year Pages
Springer-Verlaq Tokyo 2000 274-296

Author Title of Book
de Barry J. Receptor Activation Studies by Ca2+, Thermal, and PKC Imaging. in Slow Synaptic Responses and Modulation
Publisher Year Pages
Springer-Verlaq Tokyo 2000 188-199

Author Title of Book
Kojima, H. Properties of AMPA Receptor Channel During Long-Term Depression in Rat Celebellar Purkinje Cells
Publisher Year Pages
Springer-Verlaq Tokyo 2000 307-314

Author Title of Book
Allen C. N. Synaptic Transmittion and Signal Transduction in the Suprachiasmic Nucleus
Publisher Year Pages
Springer-Verlaq Tokyo 2000 386-396

Author Title of Book
Ikeda, M. Dual Imaging of Ca2+ and Cl- in the Suprachiasmatic Nucleus
Publisher Year Pages
Springer-Verlaq Tokyo 2000 200-205


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