Dynamics of Nerve Growth Cone Guidance and Adhesion

Using recent advance in光学技术we investigated functions of growth cones of culturedneurons isolated from either the central or peripheral nervous system.Inward currents were recordedfrom DRG cell bodies in response to mechanical stimuli to growth cones with the whole-cell patchclamp method at the holding potential of -60mV. Their reversal potential changed according to theNernst equation for Cl我们D1- D1.他们被completely abolished with extracellular application ofNPPB. It is concluded that the inward currents are due to activation of mechanosensitive ClD12 channels preferentially locating on the growth cones. cavisualized by using imuno-gold particles. Cultured DRG neurons pretreated with Ca D12+ D1blockers, ω-agatoxin, ω-conotoxin and nicardipine，were incubated with the respective antibody, finally with gold-conjugated antibody. Observationswith the scanning and transmission electron microscopy showed that…More ω- agatoxine - and ω- contoxine - sensitive Ca D12+ D1 channels were immunologicallylocalized on the growth cones.The [Ca - D12+ d - D1] - D2i - d -increase evoked by cell body stimulationwas inhibited by ω-agatoxin and ω-conotoxinbut not by nicardipine. The [Ca D12+ d - 1] D2i - d -increase induced by 47mm - k - d - 1+ d - 1 solutioneven in the presence of both peptide toxins was blocked by nicardipine，suggesting that nicardipine-sensitive Ca D12+ D1 channels may localize on the growthcones.Neurites in the frog spinal cord grew toward the negative electrode on untreated Falcon tissueculture plastic or on laminine substrate （negatively charged）but neurites growing on polylysine (positively charged) turned toward the positive electrode. thecharge of the growth surface influenced the frequency of anodal gavanotropism.它包含了the direction of neurite growth in an electric field is influenced by both substratum charge andgrowth cones -to substratum adhesivity.Dynamic changes of growth cones stained with DiI were observedwith a total internal反射fluorescence microscope (TIRFM). High K - 1+ High K - 1 stimulation togrowth cone regions increased DiI-TIRFM intensity in those regions，demonstrating that the basal membrane of the growth cone approached the glass substrate. the changesin the fluorescence intensity of fluo 3-loaded SCG neurons observed with the TIRFM；TIRFM images showed a pattern of spotted area of which brightness may indicate the[Ca - D12+ - D1] - D2i - D2 in submembrane space. Less

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 … More 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