Investigation of the molecular mechanism of the neurite-outgrowth using a contrast enhanced camera and intracellular perfusion methods.

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The present project has been undertaken to clarify the molecular mechanism of the neurite-outgrowth in the higher vertebrate by using a contrast enhancing CCD camera and intracellular perfusion techniques. A high-magnification video microscopy using a contrast enhanced CCD camera could detect the axoplasmic flow in the fine filopodia around the growth cone region of the cultured adult rat dorsal root ganglion neuron. The direction of the axoplasmic flow was retrograde against neurite-elongation direction in the most cases. The velocity of the retrograde axoplasmic flow in filopodia distributed between 1 to 5 mum at 28ﾟC.Biophysical properties of the ionic channels of the growth cone membrane was investigated by means of whole cell patch clamp methods. Especially, properties of Ca-channel was extensively analysed in Na^+-free, TEA-containing external solution. Cs^+ was perfused intracellularly to block K-channel. Two types of high voltage activated Ca-channels, nifedipine-sensitive L-ty … More pe Ca-channel and omega-conotoxin sensitive N-type Ca-channels, were demonstrated at the growth cone region. So far, low voltage activated T-type Ca-channel has not been demonstrated in this region. Then, effects of Ca-channel blockers on the neurite-outgrowth were examined. La^<3+> (5 muM) and nifedipine (5 muM), added in a serum-free cultured medium, suppressed sprouting and outgrowth of the neurite, but omega-conotoxin (5 muM) did not. Close relationship between nifedipine-sensitive L-type Ca-channel activity and neurite-elongation was suggested.Intracellular free Ca^<2+> concentration was measured by fluorometry using fura-2 under the presence of Ca-channel blockers. Though measured [Ca^<2+>] in of the cell body was not different between nifedipine-treated and omega-conotoxin-treated groups, "hotspots" of higher [Ca^<2+>] in were observed near the sprouting filopodia under the presence of omega-conotoxin. These results suggested close relationship between the nifedipine-sensitive Ca-channel and the neurite-sprouting. Less