哺乳動物成熟後的中樞神經系統在受到嚴重損傷後幾乎無法再生，但目前已知是可以通過外在的刺激來增強視網膜節細胞的神經活性來達到促進神經生長的目的，同時也有實驗證實利用光遺傳學的方式，通過用藍光刺激發育中小鼠視網膜節細胞上所表達的感光通道蛋白可以調控其神經活性，並達到促進視網膜神經纖維再生的目的。在本篇研究中希望探究在僅刺激ipRGCs時是否可以增強視網膜的神經纖維再生能力。結果顯示，在實驗初期使用1/60 Hz (30秒亮，30秒暗)藍光刺激ipRGCs一小時可以明顯增強P11野生型小鼠視網膜神經纖維的再生能力。雖然在發育階段的視網膜中只有非常少量的ipRGCs，但在ipRGCs和其他不同類型的RGCs之間廣泛存在著間隙連接通道，從而表現出藍光刺激後能增強整片視網膜神經纖維再生的效果。同時我們發現因為處於發育階段的小鼠，仍然存在的間隙連接通道蛋白，可以將因光刺激而產生的神經活性擴散到整片視網膜，從而使得神經纖維能大範圍的再生。同時發現在給予1/60 Hz藍光刺激的同時阻斷第二期視網膜波，會顯著的降低P11野生型小鼠視網膜的神經纖維生長。因此本實驗不僅證實ipRGCs在促進發育中的視網膜神經再生中扮演重要角色，同時還說明第二期視網膜波也在促進神經纖維再生中是不可或缺的一部分。

The mature mammalian central nervous system fails to regenerate after severe injuries. However, it is known that the increased neural activity in mouse retinal ganglion cells (RGCs) by extrinsic stimulation can promote their axon growth. Recently, it has been shown that using blue light to stimulate transgenic mice that channelrhodopsin-2 (ChR2) is expressed exclusively in RGCs can modulate neural activity and enhance neurite outgrowth of postnatal retinal explants. In the mammalian retina, there are intrinsic photosensitive retinal ganglion cells (ipRGCs), thus blue light stimulation may also activate this subset of RGCs and promote axon growth. In the present study, the aim was to examine whether activating ipRGCs alone can induce the neurite outgrowth of postnatal explants. The results showed that activation of ipRGCs by blue light stimulation with the 1/60 Hz temporal pattern (30 s “on” and 30 s “off”) for one hour at the beginning of the experiment can significantly enhance neurite outgrowth of retinal explants in P11 wild type mice. While the number of ipRGCs is low in the developing retina, the fact that there are extensive gap junction couplings among ipRGCs and other types of RGCs which allows the spread neural activity across the whole of the retina and thus manifests the effect of blue light stimulation on neurite outgrowth. It was also found that blocking the stage II retinal waves during 1/60 Hz blue light stimulation significantly reduced neurite outgrowth of retinal explants in P11 mice, suggesting that endogenous correlated waves provide the most important neural activity to the neurite outgrowth. Thus, the present findings demonstrate that ipRGCs play a key role in facilitating the neurite outgrowth of retinal explants in the developing retina and stage II retinal waves are an indispensable component in promoting neurite regeneration.

摘要 i
Abstract ii
致謝 iv
1. Introduction 1
1.1 Neural activity and axon regeneration 1
1.2 Intrinsically photosensitive retinal ganglion cells 2
1.3 Retinal waves during retinal development 3
1.4 Specific aim and brief summary 3
2. Materials and Methods 5
2.1 Animals 5
2.2 Retinal explant preparation 5
2.3 Retinal explant culture 6
2.4 Light stimulation 6
2.5 Immunohistochemistry 7
2.6 Neurite outgrowth quantification 7
2.7 Electrophysiological recording and analysis 8
2.8 Pharmacological treatment 9
2.9 Statistics 9
3. Results 11
3.1 Blue light stimulates ipRGCs and enhances neural activity of retinal explants in P11 mice 11
3.2 Blue light stimulation promotes neurite outgrowth of retinal explants in P11 mice 12
3.3 Responses of ipRGCs evoked by blue light stimulation spread across the retina via gap junctions in P11 mice. 12
3.4 Activation of ipRGCs is the major driving force to promote neurite outgrowth of retinal explants in P11 mice 13
3.5 Stage II retinal waves are important for ipRGCs driven neural activity in promoting neurite outgrowth of P11 mice 14
3.6 Activation of ipRGCs facilitates intrinsic neural activities of P11 retinal explants 15
4. Discussion 17
4.1 ipRGCs is the sole source of light activated neural activity in postnatal retinas before eye opening to promote neurite outgrowth 17
4.2 Activation of melanopsin expressing RGCs is crucial for early retinal development 19
4.3 Temporal characteristics of blue light stimulation are essential for evoking ipRGCs and enhancing neurite outgrowth 20
4.4 Glutamatergic and cholinergic neural transmissions modulate neurite outgrowth of retinal explants 21
4.5 Limitation of neurite outgrowth enhanced by increased neural activity alone 22
5. References 23
6. Figures 29

Apara A, Goldberg JL (2014) Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors. Neural Regen Res 9:1418-1421.
Arroyo DA, Kirkby LA, Feller MB (2016) Retinal waves modulate an intraretinal circuit of intrinsically photosensitive retinal ganglion cells. J Neurosci 36:6892–6905.
Bansal A, Singer JH, Hwang BJ, Xu W, Beaudet A, Feller MB (2000) Mice lacking specific nicotinic acetylcholine receptor subunits exhibit dramatically altered spontaneous activity patterns and reveal a limited role for retinal waves in forming ON and OFF circuits in the inner retina. J Neurosci 20:7672–7681.
Berkowitz BA, Roberts R, Bissig D (2010) Light-dependant intraretinal ion regulation by melanopsin in young awake and free moving mice evaluated with manganese-enhanced MRI. Mol Vis 16:1776–1780.
Berson DM (2003) Strange vision: Ganglion cells as circadian photoreceptors. Trends Neurosci 26: 314–320.
Corredor RG, Goldberg JL (2009) Electrical activity enhances neuronal survival and regeneration. J Neural Eng 6:55001.
Corredor RG, Trakhtenberg EF, Pita-Thomas W, Jin X, Hu Y, Goldberg JL (2012) Soluble adenylyl cyclase activity is necessary for retinal ganglion cell survival and axon growth. J Neurosci 32:7734–7744.
Ecker JL, Dumitrescu ON, Wong KY, Alam NM, Chen SK, LeGates T, Renna JM, Prusky GT, Berson DM, Hattar S (2010) Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision. Neuron 67:49–60.
Firth SI, Wang CT, Feller MB (2005) Retinal waves: mechanisms and function in visual system development. Cell Calcium 37:425–432.
Galli L, Maffei L (1988) Spontaneous impulse activity of rat retinal ganglion cells in prenatal life. Science 242:90–91.
Giger RJ, Hollis ER, Tuszynski MH (2010) Guidance molecules in axon regeneration. Cold Spring Harb Perspect Biol 2:a001867.
Goldberg JL (2012) Role of electrical activity in promoting neural repair. Neurosci Lett 519:134-137.
Goldberg JL, Klassen MP, Hua Y, Barres BA (2002) Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296:1860–1864.
Hansen KA, Torborg CL, Elstrott J, Feller MB (2005) Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina. J Comp Neurol 493:309–320.
Hattar S, Liao HW, Takao M, Berson DM, Yau KW (2002) Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295: 1065-1070.
He Q, Xu HP, Wang P, Tian N (2013) Dopamine D1 receptors regulate the light dependent development of retinal synaptic responses. PLoS One 8:e79625.
Hu EH, Pan F, Völgyi B, Bloomfield SA (2010) Light increases the gap junctional coupling of retinal ganglion cells. J Physiol 588:4145–4163.
Kirkby LA, Feller MB (2013) Intrinsically photosensitive ganglion cells contribute to plasticity in retinal wave circuits. Proc Natl Acad Sci USA 29:12090–12095.
Kothmann WW, Massey SC, O'Brien J (2009) Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling. J Neurosci 29:14903–14911.
Lee MJ, Chiao CC (2016) Short-term alteration of developmental neural activity enhances neurite outgrowth of retinal explants. Invest Ophthalmol Vis Sci 57:6496-6506.
Li S, Yang C, Zhang L, Gao X, Wang X, Liu W, Wang Y, Jiang S, Wong YH, Zhang Y, Liu K (2016) Promoting axon regeneration in the adult CNS by modulation of the melanopsin/GPCR signaling. Proc Natl Acad Sci USA 113:1937–1942.
Liu K, Lu Y, Lee JK, Samara R, Willenberg R, Sears-Kraxberger I, Tedeschi A, Park KK, Jin D, Cai B, Xu B, Connolly L, Steward O, Zheng B, He Z (2010) PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat Neurosci 13:1075–1081.
McLaughlin T, Torborg CL, Feller MB, O’Leary DD (2003) Retinotopic map refinement requires spontaneous retinal waves during a brief critical period of development. Neuron 40:1147–1160.
Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA (1995) Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15:805–819.
Meyer-Franke A, Wilkinson GA, Kruttgen A, Hu M, Munro E, Hanson MG, Reichardt LF, Barres BA (1998) Depolarization and cAMP elevation rapidly recruit trkB to the plasma membrane of CNS neurons. Neuron 21:681–693.
Mills SL, Xia XB, Hoshi H, Firth SI, Rice ME, Frishman LJ, Marshak DW (2007) Dopaminergic modulation of tracer coupling in a ganglion-amacrine cell network. Vis Neurosci 24:593–608.
Moore DL, Goldberg JL (2011) Multiple transcription factor families regulate axon growth and regeneration. Dev Neurobiol 71:1186–1211.
Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon VP, Goldberg JL (2009) KLF family members regulate intrinsic axon regeneration ability. Science 326:298–301.
Mrsic-Flogel TD, Hofer SB, Creutzfeldt C, Cloez-Tayarani I, Changeux JP, Bonhoeffer T, Hubener M (2005) Altered map of visual space in the superior colliculus of mice lacking early retinal waves. J Neurosci 25:6921–6928.
Muir-Robinson G, Hwang BJ, Feller MB (2002) Retinogeniculate axons undergo eye-specific segregation in the absence of eye-specific layers. J Neurosci 22:5259–5264.
Müller LP, Do MT, Yau KW, He S, Baldridge WH (2010) Tracer coupling of intrinsically photosensitive retinal ganglion cells to amacrine cells in the mouse retina. J Comp Neurol 518:4813–4824.
Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis I, Sahin M, He Z (2008) Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322:963–966.
Ramon y Cajal S (1928) Degeneration and regeneration of the nervous system. London: Oxford University Press.
Rao S, Chun C, Fan J, Kofron JM, Yang MB, Hegde RS, Ferrara N, Copenhagen DR, Lang RA (2013) A direct and melanopsin-dependent fetal light response regulates mouse eye development. Nature 494:243–246.
Reifler AN, Chervenak AP, Dolikian ME, Benenati BA, Li BY, Wachter RD, Lynch AM, Demertzis ZD, Meyers BS, Abufarha FS, Jaeckel ER, Flannery MP, Wong KY (2015) All spiking, sustained ON displaced amacrine cells receive gap-junction input from melanopsin ganglion cells. Curr Biol 25:2763–2773.
Renna JM, Weng S, Berson DM (2011) Light acts through melanopsin to alter retinal waves and segregation of retinogeniculate afferents. Nat Neurosci 14:827–829.
Schwab ME, Bartholdi D (1996) Degeneration and regeneration of axons in the lesioned spinal cord. Physiol Rev 76:319–370.
Sekaran S, Lupi D, Jones SL, Sheely CJ, Hattar S, Yau KW, Lucas RJ, Foster RG, Hankins MW (2005) Melanopsin-dependent photoreception provides earliest light detection in the mammalian retina. Curr Biol 15:1099–1107.
Sernagor E, Eglen SJ, Wong RO (2001) Development of retinal ganglion cell structure and function. Prog Retin Eye Res 20:139–174.
Stellwagen D, Shatz CJ (2002) An instructive role for retinal waves in the development of retinogeniculate connectivity. Neuron 33:357–367.
Stiles TL, Kapiloff MS, Goldberg JL (2014) The role of soluble adenylyl cyclase in neurite outgrowth. Biochim Biophys Acta 1842:2561–2568.
Syed MM, Lee S, Zheng J, Zhou ZJ (2004) Stage-dependent dynamics and modulation of spontaneous waves in the developing rabbit retina. J Physiol 560:533–549.
Vuong HE, Hardi CN, Barnes S, Brecha NC (2015) Parallel inhibition of dopamine Amacrine cells and intrinsically photosensitive retinal ganglion cells in a non-image-forming visual circuit of the mouse retina. J Neurosci 35:15955-15970.
Wang Z, Liu S, Kakizaki M, Hirose Y, Ishikawa Y, Funato H, Yanagisawa M, Yu Y, Liu Q (2014) Orexin/hypocretin activates mTOR complex 1 (mTORC1) via an Erk/Akt-independent and calcium-stimulated lysosome v-ATPase pathway. J Biol Chem 289:31950-31959.
Witkovsky P, Veisenberger E, Haycock JW, Akopian A, Garcia-Espana A, Meller E (2004) Activity-dependent phosphorylation of tyrosine hydroxylase in dopaminergic neurons of the rat retina. J Neurosci 24:4242–4249.