Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
Neuroscience in general
|Research Institution||Tokyo Medical and Dental University|
KATAYAMA Yoshifumi Tokyo Med & Dent Univ, Med Res Inst, Prof, 難治疾患研究所, 教授 (20014144)
TATSUMI Hitoshi Nagoya Univ, Sch Med Assit Prof, 医学部, 助手 (20171720)
FURUKAWA Tetsushi Tokyo Med & Dent Univ, Med Res Inst, Assist Prof, 難治疾患研究所, 助手 (80251552)
HIRAI Kenji Tokyo Med & Dent Univ, Med Res Inst, Assoc Prof, 難治疾患研究所, 助教授 (70156628)
MCCAING C.D. Univ Aberdeen, Biomed Sci, Senior Lecturer, 医学部, 上級講師
TSUJI S. P. &M. キュリー大学, CNRS, 教授
TSUJI Shigeru P & M Curie Univ, CNRS,Research Director
MCCAIG C. D. アバディーン大学, 医学部, 上級講師
MCCAIG C.D. アバディーン大学, 医学部, 準教授
|Project Period (FY)
1996 – 1997
Completed(Fiscal Year 1997)
|Budget Amount *help
¥8,600,000 (Direct Cost : ¥8,600,000)
Fiscal Year 1997 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Fiscal Year 1996 : ¥4,300,000 (Direct Cost : ¥4,300,000)
|Keywords||Neuron / Growth cone / Synapse / Neurotransmitter / Optical technology / Ion channel / Electric field / Mechanosensitivity / 光学技術 / 光ピンセット / 近接場光学顕微鏡 / カルシウムチャネル / 神経成長円錐 / 神経伝達物質 / カルシウム / 神経成長因子 / ホールセルパッチクランプ|
It is necessary to apply recent advance in optical technology to studying cellular physiology. Using some new techniques, we demonstrated that growth cones of rat cultured neurons can release neurotransmitter and be sensitive to both mechanical and electric field stimulation.
Glutamate release from growth cones was detected by using an acutely dissociated hippocampal neuron as a biodetector from which whole cell patch clamp recordings were made. The release was dependent on Ca^<2+> and was abolished by omega-agatoxin. Cultured neurons were pretreated with the toxin, then incubated with the antibody to the toxin and further stained with gold-conjugated antibody. Since clusters of immuno-gold particles were observed over the membrane of growth cones by means of scanning and transmission electron microscpes, it is suggested that omega-agatoxin-sensitive Ca^<2+> channels were immunologically localized on growth cones.
The mechanism underlying cathodally-directed growth of cultured DRG neurit
es was investigated by using confocal laser scanning microscopy to observe cytoskeletal elements, second messenger signaling elements and the nucleus in neurons exposed to small physiological electric fields. Three types of fluorescent probes were used, a) Rhodamine halloidin stains filamentous actin, b) a fluorescein-conjugated monoclonal antibody labelled proteins phosphorylated on tyrosine residues and c) a marker for mitochondria clearly delineated the position of the nucleus by exclusion, being present only in the cytoplasma. Physiological electric fields induce asymmetries of the filamentous actin cytoskeleton and proteins phosphorylated on tyrosine residues in growth cones, and alter the positioning of cell nucleus.
When mechanical stimulation was applied to growth cones of DRG neurons, inward currents were recorded, from their soma at the holding potential of -60mV,using whole cell patch clamp technique. Almost no electrical response was recorded when other sites except lamellipodium were stimulated. The outward currents were supposed to be due mainly to activation of Cl^- channels according to their electrophysiological properties.
Evanescent light illumination was introduced into a high resolution video-enhanced differential interference contrast microscope. Growth cones and soma of neurons which were cultured on glass plates were stained with a fluorescent dye, Dil, and were observed with an internal total reflection evanescent light microscope. The evanescent images showed that growth cones and some limited areas of soma attached to the surface of culture glass plate. Less