It is known that guidance cues play important roles in neuronal outgrowth and axonal path-finding. These guidance cues, when bound to the receptors on nerve growth cones, trigger a series of signal transduction pathways that result in dynamic rearrangement of cytoskeleton in neuronal cells. Although enormous studies have been suggested that intracellular calcium concentration is crucial in regulation of neurite outgrowth and cytoskeleton rearrangement, the precise molecular mechanism underlying growth cone guidance is not well understood. The aim of our studies is to set up an evaluation system, growth cone turning assay, which is suitable and reproducible for exploration of the molecular mechanism of guidance cue-induced growth cone turning. The 1-day-old cultured spinal neurons of Xenopus laevis and the approved attractive guidance cue, cAMP, were used in our growth cone turning assay.
We evaluate the effects of neuronal developmental stage, size of pressure, frequency and duration in cAMP-induced growth cone turning. We found application of cAMP with a pressure of 2 Hz in frequency and 20 ms in duration can elicit a reliable growth cone attractive response in cultured spinal motoneurons with 14~18 hours in age. Depletion of intracellular calcium store with thapsigargin in calcium-free Ringer could significantly abolish cAMP-induced growth cone turning. The cAMP-induced growth cone turning was not blunted when calcium was omitted from extracellular fluid or bath application of calcium channel inhibitor suggesting calcium influx is not responsible for the growth cone attractive effect of cAMP. Application of membrane-permeable inhibitors of ryanodine receptors but not inositol 1,4,5-trisphosphate (IP3) effectively occluded the attractive effect elicited by cAMP. The cAMP-induced growth cone turning was unapparent under the presence of Li+, a blocker of IP3 formation. It has been suggest besides block IP3 formation, Li+ is also involved in modulation of several signaling pathways including GSK3-£] dependent signaling. However, bath application of GSK3-£] inhibitor has no significant effect on cAMP-induced growth cone turning.
Taken together, our results suggest ryanodine-mobilized intracellular Ca2+ store play a major role in cAMP-induced growth cone turning. Moreover, our result on surveying various embryonic stage, drug application protocol also establish a good condition for neuronal growth cone turning assay, which can be used in exploring the molecular mechanism of growth cone path-finding.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0616106-154849 |
| Date | 16 June 2006 |
| Creators | Jiang, kun-ci |
| Contributors | none, none, none |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0616106-154849 |
| Rights | withheld, Copyright information available at source archive |
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