H2O2 為一種可穿透細胞膜的活性氧化物 (ROS)，它可經由細胞內粒線體呼吸作用、細胞上 NADPH oxidase、嘌呤 (purine) 代謝酵素 xanthine oxidase 作用中生成。H2O2 較人為常知的是它對於細胞毒性的部分，例如 DNA 損傷、細胞膜的過氧化、細胞老化等，然而其正常生理角色的研究較為少。在本實驗中，我們利用發育中爪蟾神經-肌突觸細胞培養，以 whole-cell patch clamp 方式記錄 H2O2 於自發性突觸電流 (SSCs) 的影響。實驗結果發現，以抗氧化劑 NAC 和 Sodium pyruvate 處理可降低神經傳遞物質的釋放，顯示內生性 H2O2 在神經傳遞物質釋放上扮演重要角色。在 culture 中，外給不可穿透細胞膜的H2O2 scavenger－catalase 時並不影響神經活性，然而以 catalase 送至肌細胞將胞內 H2O2 清除時，神經活性略微降低，此時即使再外給 H2O2 亦不見促進作用，顯示其作用並非由突觸後肌細胞產生 H2O2 後釋放至胞外直接影響神經末梢釋放神經傳遞物質 (retrograde) 或突觸前神經細胞產生後釋放出來影響肌細胞 ACh 接受器之敏感度，而是在肌細胞內產生H2O2 間接影響神經活性。
當我們給予分別粒線體 electron transport chain complex I 及 complex III 抑制劑 rotenone 與 antimycin 時會提高神經活性。而以 NADPH oxidase 抑制劑 (DPI、apocynin、AEBSF) 處理可見增加神經
v
傳遞物質釋放頻率或減少振幅，此結果與預期有所出入而有待後續實驗探討。而以 xanthine oxidase 抑制劑 allopurinol 處理可見有意義的少許減少的情形，因此，肌細胞內影響神經活性的 H2O2 可能是由粒線體及 xanthine oxidase 生成而來。
已有文獻表示，H2O2 會誘導胞內鈣離子釋放，神經傳遞物質釋放時，胞內鈣離子濃度上升是必要的過程，當以不可穿膜的鈣離子螯合劑 BAPTA 降低肌細胞內鈣離子濃度後再給予 H2O2，發現可降低 H2O2 的促進作用，顯示肌細胞內 Ca2+ 的存在與 H2O2 後續促使神經傳遞物質釋放增加有關。在胞內 H2O2 可修飾蛋白影響其活性以及活化基因表現、蛋白質合成等。而 H2O2 在肌細胞如何影響神經活性的探討，實驗中以蛋白質合成抑制劑 anisomycin 和 cycloheximide 處理結果並不會影響 H2O2 的促進作用，顯示 H2O2 造成的促進作用並非經由誘發合成蛋白質來影響神經釋放神經傳遞物質。已知神經滋養因子 IGF-1 在突觸發育階段能夠促進神經傳遞物質釋放，因此以 IGF-1 receptor 阻斷劑 (JB-1) 將 receptor 阻斷掉，發現能夠降低 H2O2 的促進作用。由以上結果推斷，由肌細胞內粒線體和 xanthine oxidase 生成的 H2O2 經由胞內 Ca2+ 增加，並誘發 IGF-1 等滋養因子釋放，進而促進神經傳遞物質釋放。

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
vii
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
viii
our studies have shed some light on the molecular mechanism of the formation of developing neuromuscular synapse.

目 錄
縮寫表 .................................................................................................... iii
中文摘要 ................................................................................................ iv
英文摘要 ................................................................................................ vi
緒論 ........................................................................................................ 1
實驗材料 ................................................................................................ 13
實驗方法 ................................................................................................ 16
§ 電生理紀錄方法 ..................................................................... 16
結果 ........................................................................................................ 19
討論 ........................................................................................................ 28
參考文獻 ................................................................................................ 37
圖表 ........................................................................................................ 43

Ψ參考文獻
Anderson, M.J., Cohen, M.W. & Zorychta, E. (1977) Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol, 268, 731-756.
Auerbach, J.M. & Segal, M. (1997) Peroxide modulation of slow onset potentiation in rat hippocampus. J Neurosci, 17, 8695-8701.
Banker, G. & Goslin, K. (1998) Culturing nerve cells. MIT Press, Cambridge, Mass.
Bao, L., Avshalumov, M.V., Patel, J.C., Lee, C.R., Miller, E.W., Chang, C.J. & Rice, M.E. (2009) Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling. J Neurosci, 29, 9002-9010.
Barallobre, M.J., Pascual, M., Del Rio, J.A. & Soriano, E. (2005) The Netrin family of guidance factors: emphasis on Netrin-1 signalling. Brain Res Brain Res Rev, 49, 22-47.
Bleau, G., Giasson, C. & Brunette, I. (1998) Measurement of hydrogen peroxide in biological samples containing high levels of ascorbic acid. Anal Biochem, 263, 13-17.
Cao, G. & Ko, C.P. (2007) Schwann cell-derived factors modulate synaptic activities at developing neuromuscular synapses. J Neurosci, 27, 6712-6722.
Chance, B., Sies, H. & Boveris, A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev, 59, 527-605.
Dalle-Donne, I., Rossi, R., Milzani, A., Di Simplicio, P. & Colombo, R. (2001) The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself. Free Radic Biol Med, 31, 1624-1632.
Dalton, T.P., Shertzer, H.G. & Puga, A. (1999) Regulation of gene
38
expression by reactive oxygen. Annu Rev Pharmacol Toxicol, 39, 67-101.
Deby, C. & Goutier, R. (1990) New perspectives on the biochemistry of superoxide anion and the efficiency of superoxide dismutases. Biochem Pharmacol, 39, 399-405.
Delafontaine, P. & Ku, L. (1997) Reactive oxygen species stimulate insulin-like growth factor I synthesis in vascular smooth muscle cells. Cardiovasc Res, 33, 216-222.
Droge, W. (2002) Free radicals in the physiological control of cell function. Physiol Rev, 82, 47-95.
Fiaschi, T., Cozzi, G., Raugei, G., Formigli, L., Ramponi, G. & Chiarugi, P. (2006) Redox regulation of beta-actin during integrin-mediated cell adhesion. J Biol Chem, 281, 22983-22991.
Gautam, D.K., Misro, M.M., Chaki, S.P. & Sehgal, N. (2006) H2O2 at physiological concentrations modulates Leydig cell function inducing oxidative stress and apoptosis. Apoptosis, 11, 39-46.
Giambelluca, M.S. & Gende, O.A. (2008) Hydrogen peroxide activates calcium influx in human neutrophils. Mol Cell Biochem, 309, 151-156.
Giannoni, E., Buricchi, F., Raugei, G., Ramponi, G. & Chiarugi, P. (2005) Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol Cell Biol, 25, 6391-6403.
Giniatullin, A.R. & Giniatullin, R.A. (2003) Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction. J Physiol, 552, 283-293.
Gomez-Cabrera, M.C., Close, G.L., Kayani, A., McArdle, A., Vina, J. & Jackson, M.J. (2010) Effect of xanthine oxidase-generated extracellular superoxide on skeletal muscle force generation. Am J
39
Physiol Regul Integr Comp Physiol, 298, R2-8.
Granados, M.P., Salido, G.M., Gonzalez, A. & Pariente, J.A. (2006) Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells. Biochem Cell Biol, 84, 39-48.
Gunter, T.E., Buntinas, L., Sparagna, G., Eliseev, R. & Gunter, K. (2000) Mitochondrial calcium transport: mechanisms and functions. Cell Calcium, 28, 285-296.
Ishii, D.N. (1989) Relationship of insulin-like growth factor II gene expression in muscle to synaptogenesis. Proc Natl Acad Sci U S A, 86, 2898-2902.
Jackson, M.J. (2011) Control of reactive oxygen species production in contracting skeletal muscle. Antioxid Redox Signal, 15, 2477-2486.
Jagtap, J.C., Chandele, A., Chopde, B.A. & Shastry, P. (2003) Sodium pyruvate protects against H(2)O(2) mediated apoptosis in human neuroblastoma cell line-SK-N-MC. J Chem Neuroanat, 26, 109-118.
Kemmerling, U., Munoz, P., Muller, M., Sanchez, G., Aylwin, M.L., Klann, E., Carrasco, M.A. & Hidalgo, C. (2007) Calcium release by ryanodine receptors mediates hydrogen peroxide-induced activation of ERK and CREB phosphorylation in N2a cells and hippocampal neurons. Cell Calcium, 41, 491-502.
Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U. & Nishizuka, Y. (1997) Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci U S A, 94, 11233-11237.
Konishi, H., Yamauchi, E., Taniguchi, H., Yamamoto, T., Matsuzaki, H., Takemura, Y., Ohmae, K., Kikkawa, U. & Nishizuka, Y. (2001) Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro. Proc Natl Acad Sci U S A, 98, 6587-6592.
40
Kress, M., Riedl, B. & Reeh, P.W. (1995) Effects of oxygen radicals on nociceptive afferents in the rat skin in vitro. Pain, 62, 87-94.
Li, C. & Jackson, R.M. (2002) Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol, 282, C227-241.
Liu, Y., Fiskum, G. & Schubert, D. (2002) Generation of reactive oxygen species by the mitochondrial electron transport chain. J Neurochem, 80, 780-787.
Los, M., Droge, W., Stricker, K., Baeuerle, P.A. & Schulze-Osthoff, K. (1995) Hydrogen peroxide as a potent activator of T lymphocyte functions. Eur J Immunol, 25, 159-165.
Markadieu, N., Crutzen, R., Blero, D., Erneux, C. & Beauwens, R. (2005) Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers. Am J Physiol Renal Physiol, 288, F1201-1212.
Massaad, C.A. & Klann, E. (2011) Reactive oxygen species in the regulation of synaptic plasticity and memory. Antioxid Redox Signal, 14, 2013-2054.
Mattson, M.P. (2005) NF-kappaB in the survival and plasticity of neurons. Neurochem Res, 30, 883-893.
Mbong, N. & Anand-Srivastava, M.B. (2012) Hydrogen peroxide enhances the expression of Gialpha proteins in aortic vascular smooth cells: role of growth factor receptor transactivation. Am J Physiol Heart Circ Physiol, 302, H1591-1602.
Mittal, C.K. & Murad, F. (1977) Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3',5'-monophosphate formation. Proc Natl Acad Sci U S A, 74, 4360-4364.
41
Mohazzab, K.M., Kaminski, P.M. & Wolin, M.S. (1994) NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol, 266, H2568-2572.
Munnamalai, V. & Suter, D.M. (2009) Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth. J Neurochem, 108, 644-661.
Murphy, M.P. (2009) How mitochondria produce reactive oxygen species. Biochemical Journal, 417, 1-13.
Ragan, C.I. & Bloxham, D.P. (1977) Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium. Biochem J, 163, 605-615.
Rao, S.S., Stewart, B.A., Rivlin, P.K., Vilinsky, I., Watson, B.O., Lang, C., Boulianne, G., Salpeter, M.M. & Deitcher, D.L. (2001) Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant. EMBO J, 20, 6761-6771.
Servitja, J.M., Masgrau, R., Pardo, R., Sarri, E. & Picatoste, F. (2000) Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices. J Neurochem, 75, 788-794.
Silveira, L.R., Pereira-Da-Silva, L., Juel, C. & Hellsten, Y. (2003) Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radic Biol Med, 35, 455-464.
Soto, F., Ma, X., Cecil, J.L., Vo, B.Q., Culican, S.M. & Kerschensteiner, D. (2012) Spontaneous activity promotes synapse formation in a cell-type-dependent manner in the developing retina. J Neurosci, 32, 5426-5439.
Spitzer, N.C. & Lamborghini, J.E. (1976) The development of the action potential mechanism of amphibian neurons isolated in culture. Proc Natl Acad Sci U S A, 73, 1641-1645.
42
Tretter, L. & Adam-Vizi, V. (1996) Early events in free radical-mediated damage of isolated nerve terminals: effects of peroxides on membrane potential and intracellular Na+ and Ca2+ concentrations. J Neurochem, 66, 2057-2066.
Tsentsevitsky, A., Nikolsky, E., Giniatullin, R. & Bukharaeva, E. (2011) Opposite modulation of time course of quantal release in two parts of the same synapse by reactive oxygen species. Neuroscience, 189, 93-99.
Weiss, S.J. (1986) Oxygen, ischemia and inflammation. Acta Physiol Scand Suppl, 548, 9-37.
White, A.A., Crawford, K.M., Patt, C.S. & Lad, P.J. (1976) Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide. J Biol Chem, 251, 7304-7312.
Zhong, J. & Lee, W.H. (2007) Hydrogen peroxide attenuates insulin-like growth factor-1 neuroprotective effect, prevented by minocycline. Neurochem Int, 51, 398-404.