在成熟的哺乳類動物中，中樞神經系統若受到嚴重損傷便無法再生。然而，目前已知利用外在的刺激來提升視網膜節細胞的神經活性可促進軸突的生長。在本篇研究中，我們採用光遺傳學的方式來探索是否可利用藍光刺激來調控發育中小鼠視網膜節細胞中表現感光通道蛋白的神經活性，進而促進其視網膜的神經纖維再生。結果顯示，利用一小時的20 Hz藍光刺激來活化帶有感光通道蛋白的視網膜節細胞最能有效地增強視網膜的神經纖維再生，且這是藉由細胞間的間隙連接來將藍光刺激所引起的神經活性傳遞到整片視網膜來達成大範圍的視網膜節細胞神經再生。再者，我們也發現藉由藍光而活化的感光視網膜節細胞同時也扮演著促進發育中視網膜神經纖維再生的角色。這些發現不只證明了短期地增加視網膜節細胞的神經活性就足以促進視網膜的神經再生，同時也點出了利用具備時間特性的外在刺激來調控節細胞的神經活性是一個關鍵且有效的方式來促使視神經再生。

Neurons in the adult mammalian CNS fails to regenerate after severe injury. However, it is known that an increase in neural activity occurs in mouse retinal ganglion cells (RGCs) after extrinsic stimulation and this can induce axon growth. In the present study, we applied an optogenetic approach using a mouse model specifically involving channelrhodopsin-2 (ChR2) expression in RGCs. We investigated whether modulation of RGC neural activity exclusively by blue light stimulation is able to promote neurite outgrowth of postnatal retinal explants. The results showed that activation by 20 Hz blue light for one hour of RGCs expressing ChR2 is a most effective way of enhancing neurite outgrowth in postnatal retinas. This is achieved via gap junctions that spread neural activity across the whole of the retina. Moreover, we found that the activation of intrinsically photosensitive retinal ganglion cells by blue light also contributes significantly to the promotion of neurite outgrowth in the same postnatal retinal explants. Our findings not only demonstrate that a short-term increase in RGC neural activity is sufficient to facilitate the neurite outgrowth of retinal explants, but also highlight the fact that the temporal pattern of neural activity in RGCs is a critical factor in regulating axon regeneration.

Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, Augustine GJ, Ehlers MD, Feng G (2007) In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54:205-218.
Arroyo DA, Kirkby LA, Feller MB (2016) Retinal waves modulate an intraretinal circuit of intrinsically photosensitive retinal ganglion cells. J Neurosci 36:6892-6905.
Berson DM, Dunn FA, Takao M (2002) Phototransduction by retinal ganglion cells that set the circadian clock. Science 295:1070-1073.
Birngruber R, Gabel VP, Hillenkamp F (1983) Experimental studies of laser thermal retinal injury. Health Phys 44:519-531.
Bradke F, Fawcett JW, Spira ME (2012) Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nat Rev Neurosci 13:183-193.
Britt JP, McDevitt RA, Bonci A (2012) Use of channelrhodopsin for activation of CNS neurons. Curr Protoc Neurosci 58:2.16. 11-12.16. 19.
Buonanno A, Fields RD (1999) Gene regulation by patterned electrical activity during neural and skeletal muscle development. Curr Opin Neurobiol 9:110-120.
Corredor RG, Goldberg JL (2009) Electrical activity enhances neuronal survival and regeneration. J Neural Eng 6:055001.
Corredor RG, Trakhtenberg EF, Pita-Thomas W, Jin XL, Hu Y, Goldberg JL (2012) Soluble adenylyl cyclase activity is necessary for retinal ganglion cell survival and axon growth. J Neurosci 32:7734-7744.
Deisseroth K (2011) Optogenetics. Nat Methods 8:26-29.
Del Olmo‐Aguado S, Manso AG, Osborne NN (2012) Light might directly affect retinal ganglion cell mitochondria to potentially influence function. Photochem. Photobiol 88:1346-1355.
Goldberg JL, Barres BA (2000) The relationship between neuronal survival and regeneration. Annu Rev Neurosci 23:579-612.
Goldberg JL, Espinosa JS, Xu Y, Davidson N, Kovacs GT, Barres BA (2002) Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron 33:689-702.
Hansen KA, Torborg CL, Elstrott J, Feller MB (2005) Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina. Comp Neurol 493:309-320.
Kirkby LA, Feller MB (2013) Intrinsically photosensitive ganglion cells contribute to plasticity in retinal wave circuits. Proc Natl Acad Sci U S A 110:12090-12095.
Leaver SG, Cui Q, Plant GW, Arulpragasam A, Hisheh S, Verhaagen J, Harvey AR (2006) AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells. Gene Ther 13:1328-1341.
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.
Lever IJ, Bradbury EJ, Cunningham JR, Adelson DW, Jones MG, McMahon SB, Marvizon JCG, Malcangio M (2001) Brain-derived neurotrophic factor is released in the dorsal horn by distinctive patterns of afferent fiber stimulation. J Neurosci 21:4469-4477.
Lim JH, Stafford BK, Nguyen PL, Lien BV, Wang C, Zukor K, He Z, Huberman AD (2016) Neural activity promotes long-distance, target-specific regeneration of adult retinal axons. Nat Neurosci 19:1073-1084.
Liu K, Tedeschi A, Park KK, He Z (2011) Neuronal intrinsic mechanisms of axon regeneration. Annu Rev Neurosci 34:131-152.
Masland RH (2012) The neuronal organization of the retina. Neuron 76:266-280.
Meyer-Franke A, Kaplan MR, Pfieger 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, Jr., Reichardt LF, Barres BA (1998) Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron 21:681-693.
Morimoto T, Miyoshi T, Matsuda S, Tano Y, Fujikado T, Fukuda Y (2005) Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest Ophthalmol Vis Sci 46:2147-2155.
Muñoz MA, Pacheco A, Becker MI, Silva E, Ebensperger R, Garcia AM, De Ioannes AE, Edwards AM (2011) Different cell death mechanisms are induced by a hydrophobic flavin in human tumor cells after visible light irradiation. Photochem Photobiol B 103:57-67.
Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A 100:13940-13945.
Ou YT, Lu MS, Chiao CC (2012) The effects of electrical stimulation on neurite outgrowth of goldfish retinal explants. Brain Res 1480:22-29.
Pérez de Sevilla Müller L, Do MTH, 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.
Park S, Koppes RA, Froriep UP, Jia X, Achyuta AK, McLaughlin BL, Anikeeva P (2015) Optogenetic control of nerve growth. Sci Rep 5:9669.
Ramon y Cajal S (1928) Degeneration and regeneration of the nervous system. London: Oxford UP.
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 (2015) All spiking, sustained ON displaced amacrine cells receive gap-junction input from melanopsin ganglion cells. Curr Biol 25:2763-2773.
Renna JM, Weng SJ, Berson DM (2011) Light acts through melanopsin to alter retinal waves and segregation of retinogeniculate afferents. Nat Neurosci 14:827-829.
Schmidt TM, Do MT, Dacey D, Lucas R, Hattar S, Matynia A (2011) Melanopsin-positive intrinsically photosensitive retinal ganglion cells: from form to function. J Neurosci 31:16094-16101.
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.
Sexton TJ, Bleckert A, Turner MH, Van Gelder RN (2015) Type I intrinsically photosensitive retinal ganglion cells of early post-natal development correspond to the M4 subtype. Neural Dev 10:17.
Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146-156.
Tarttelin EE, Bellingham J, Bibb LC, Foster RG, Hankins MW, Gregory-Evans K, Gregory-Evans CY, Wells DJ, Lucas RJ (2003) Expression of opsin genes early in ocular development of humans and mice. Exp Eye Res 76:393-396.
Thyagarajan S, van Wyk M, Lehmann K, Lowel S, Feng G, Wassle H (2010) Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells. J Neurosci 30:8745-8758.
Ting JT, Feng G (2013) Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: progress and prospects for behavioral neuroscience. Behav Brain Res 255:3-18.
Venugopalan P, Wang Y, Nguyen T, Huang A, Muller KJ, Goldberg JL (2016) Transplanted neurons integrate into adult retinas and respond to light. Nat Commun 7:10472.
Wang L, Dong J, Cull G, Fortune B, Cioffi GA (2003) Varicosities of intraretinal ganglion cell axons in human and nonhuman primates. Invest Ophthalmol Vis Sci 44:2-9.
West AE, Chen WG, Dalva MB, Dolmetsch RE, Kornhauser JM, Shaywitz AJ, Takasu MA, Tao X, Greenberg ME (2001) Calcium regulation of neuronal gene expression. Proc Natl Acad Sci 98:11024-11031.
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.
Yoshii T, Ahmad M, Helfrich-Forster C (2009) Cryptochrome mediates light-dependent magnetosensitivity of Drosophila's circadian clock. PLoS Biol 7:e1000086.
Youssef PN, Sheibani N, Albert DM (2011) Retinal light toxicity. Eye (Lond) 25:1-14.
Zhang JY, Ackman JB, Xu HP, Crair MC (2012) Visual map development depends on the temporal pattern of binocular activity in mice. Nat Neurosci 15:298-307.