1999 Fiscal Year Final Research Report Summary
Dynamics of Nerve Growth Cone Guidance and Adhesion
| Project/Area Number |
10044248
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neuroscience in general
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
KATAYAMA Yoshifumi Tokyo Med & Dent Univ, Med Res Inst, Prof, 難治疾患研究所, 教授 (20014144)
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| Co-Investigator(Kenkyū-buntansha) |
FURUKAWA Tetsushi Akita Univ, Sch Med, Assoc Prof, 医学部, 助教授 (80251552)
TATSUMI Hitoshi Nagoya Univ, Sch Med, Assoc Prof, 医学部, 助教授 (20171720)
HIRAI Keiji Tokyo Med & Dent Univ, Med Res Inst, Assoc Prof, 難治疾患研究所, 助教授 (70156628)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Nerve growth cone / Chloride channel / Calcium channel / Calcium concentration / Mechanosensitivity / Galvanotropism / Adhesivity / Transmitter release |
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| Research Abstract |
Using recent advance in optical technology we investigated functions of growth cones of cultured neurons isolated from either the central or peripheral nervous system.
Inward currents were recorded from DRG cell bodies in response to mechanical stimuli to growth cones with the whole-cell patch clamp method at the holding potential of -60mV. Their reversal potential changed according to the Nernst equation for ClィイD1-ィエD1. They were completely abolished with extracellular application of NPPB. It is concluded that the inward currents are due to activation of mechanosensitive ClィイD1-ィエD1 channels preferentially locating on the growth cones.
CaィイD12+ィエD1 channels on the growth cones were visualized by using imuno-gold particles. Cultured DRG neurons pretreated with CaィイD12+ィエD1 blockers, ω-agatoxin, ω-conotoxin and nicardipine, were incubated with the respective antibody and finally with gold-conjugated antibody. Observations with the scanning and transmission electron microscopy showed that
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ω-agatoxin- and ω-contoxin- sensitive CaィイD12+ィエD1 channels were immunologically localized on the growth cones.
The [CaィイD12+ィエD1]ィイD2iィエD2-increase evoked by cell body stimulation was inhibited by ω-agatoxin and ω-conotoxin, but not by nicardipine. The [CaィイD12+ィエD1]ィイD2iィエD2-increase induced by 47mM-KィイD1+ィエD1 solution even in the presence of both peptide toxins was blocked by nicardipine, suggesting that nicardipine-sensitive CaィイD12+ィエD1 channels may localize on the growth cones.
Neurites in the frog spinal cord grew toward the negative electrode on untreated Falcon tissue culture plastic or on laminine substrate (negatively charged), but neurites growing on poly-lysine (positively charged) turned toward the positive electrode. The charge of the growth surface influenced the frequency of anodal gavanotropism. It is concluded that the direction of neurite growth in an electric field is influenced by both substratum charge and growth cone-to substratum adhesivity.
Dynamic changes of growth cones stained with DiI were observed with a total internal reflection fluorescence microscope (TIRFM). High KィイD1+ィエD1 stimulation to growth cone regions increased DiI-TIRFM intensity in those regions, demonstrating that the basal membrane of the growth cone approached the glass substrate. The changes in the fluorescence intensity of fluo 3-loaded SCG neurons were observed with the TIRFM ; TIRFM images showed a pattern of spotted area of which brightness may indicate the [CaィイD12+ィエD1]ィイD2iィエD2 in submembrane space. Less
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