Hydrogen peroxide (H2O2), a membrane-permeable reactive oxygen species, is continuously produced by mitochondrial respiration, the membrane-associated NADPH oxidase complex, xathine oxidase catalyzed reaction. Although the cytotoxic effect of H2O2 is well documented, the role of H2O2 in synapse formation if still in its infancy. Here we test the role of H2O2 on the frequency of spontaneous synaptic currents (SSCs) at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of H2O2 dose-dependently enhances the frequency of spontaneous synaptic currents (SSC frequency). Treatment of the culture with membrane-permeable antioxidants N-acetylcysteine and sodium pyruvate significantly decreased SSC frequency, indicating endogenous reactive oxygen species play important roles in the regulation of spontaneous ACh release. Bath application of membrane non-permeable catalase, which breaks down H2O2 specifically, has no significant effect on SSC frequency, suggesting H2O2 is not an intercellular signaling molecule being produced and released from postsynaptic myocyte and affects the neurotransmitter release of presynaptic motoneuron. Much to our surprise is that the SSC frequency was significantly decreased while catalase was
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loaded into the myocyte through recording pipette. Furthermore, the SSC frequency facilitation induced by exogenously applied H2O2 was completely hampered while catalase was loaded into the myocyte. These results indicate although endogenous H2O2 in myocyte plays a crucial role on SSC frequency facilitation, this facilitation on the neurotransmitter release of presynaptic motoneuron is achieved through a retrograde factor other than H2O2 itself.
Treatment of the culture with inhibitor of either NADPH oxidase does not have significant effect on SSC frequency. Bath application of mitochondria complex I, II and xanthine oxidase inhibitor significantly decreased SSC frequency, suggesting H2O2 derived from xanthine oxidase and mitochondria is responsible for the regulation of SSC frequency. Bath application of translation blocker anisomycin and cycloheximide could not attenuate the facilitation of H2O2. Addition of IGF-1 receptor inhibitor JB-1 to the culture significantly attenuated SSC frequency. Overall, our current results suggest that xanthine oxidase activity-derived H2O2 in myocyte induce the release of IGF-1 which retrogradely enhance the spontaneous neurotransmitter release from presynaptic motoneuron. Since synaptic activity is crucial in synaptogenesis and synapse maturation, results form
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our studies have shed some light on the molecular mechanism of the formation of developing neuromuscular synapse.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0724112-155837 |
| Date | 24 July 2012 |
| Creators | Lin, Shu-Hui |
| Contributors | Chun-Lin Chen, Bin-Nan Wu, Jau-Cheng Liou |
| 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-0724112-155837 |
| Rights | user_define, Copyright information available at source archive |
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