發育中視網膜的自發週期性神經活性對於神經網絡連接和左右眼在腦內特定分層的成熟是必要的。先前的研究指出第二期視網膜波與光環境之間有相關性，但其影響和機制的細節仍然有許多未知。本篇研究中，我們發現了長時間光線刺激可以調控乙烯膽鹼相關視網膜波。在長時間光照下，視網膜波減小了波間時間間隔，並且高度相關性的細胞在空間分布上會縮減範圍大小。我們還發現在環境光刺激之後，c-fos蛋白表達神經元出現在視網膜內核層 (INL)，但星狀無軸突細胞 (SACs)很少有c-fos表達細胞存在。此外，藥物相關實驗指出，光激發的視網膜波特性變化與多巴胺和麩氨酸相關神經迴路有關連性。綜和以上這些結果，我們指出在第二期視網膜波會受到光線環境調控，而這個現象在發育中小鼠視網膜中受到多巴胺相關神經訊息所控制。

The spontaneous periodic activity in the developing retina is necessary for the maturation of neural network connections and eye-specific segregation. The previous studies demonstrate that there is a correlation between the Stage II retinal waves and the light environment, but the details of the effects and mechanisms are still largely unknown. Here, we describe about how the long-term light stimulation modulates the cholinergic waves. Under the long-term light exposure, the retinal waves reduced the inter-wave intervals and converged their spatial distribution within the highly connected channels. We also showed that the c-fos expressive neurons occurred in the inner nuclear layer after the ambient light stimulation, but the c-fos positive cells rarely co-existed with starburst amacrine cells. Moreover, the pharmacological experiments revealed that light-induced wave property changes were correlated with the dopaminergic and glutamatergic associated pathways. Together, these results support that the Stage II retinal waves are regulated by light environment and this process requires the dopaminergic signals in the developing mouse retina.

摘要 2
Abstract 3
致謝 4
Contents 6
Chapter 1. Introduction 8
1.1 The fundamental process of neural development 8
1.2 Retinal waves 9
1.3 The functions of third photosensitive neurons in mammal retinas 9
1.4 The effect of light during developmental stage 10
1.5 Aims of the present study 11
Chapter 2. Materials and methods 12
2.1 Animals and tissue preparation 12
2.2 Light stimulation 13
2.3 Electrophysiological recordings 13
2.4 Pharmacology 14
2.5 Immunohistochemistry 14
2.6 c-Fos expression quantification 15
2.7 Wave events identification and analysis 16
2.8 Statistical analysis 17
Chapter 3. Results 18
3.1 Long-term light stimulation modulates cholinergic waves in the mouse retina 18
3.2 Long-term light stimulation does not induce the c-fos expression in starburst amacrine cells 20
3.3 The light-induced wave property changes are modulated by dopaminergic signaling 21
3.4 The light-induced wave property changes are up-regulated in the presence of glutamate receptor antagonists 22
3.5 The light-induced wave property changes correlate with the dopaminergic and glutamatergic signaling pathways 24
Chapter 5. Discussion 25
4.1 The links between ipRGCs and retinal waves during the developmental stage 25
4.2 The light responses convey the excitatory signals to upstream neurons in the developing retina 27
4.3 The light transmission process between ipRGCs and dopaminergic amacrine cells in the developing retina 28
References 30
Tables 36
Figures 37

Allen, A.E., Storchi, R., Martial, F.P., Petersen, R.S., Montemurro, M.A., Brown, T.M., and Lucas, R.J. (2014). Melanopsin-driven light adaptation in mouse vision. Current biology 24, 2481-2490.
Arroyo, D.A., Kirkby, L.A., and Feller, M.B. (2016). Retinal waves modulate an intraretinal circuit of intrinsically photosensitive retinal ganglion cells. Journal of Neuroscience 36, 6892-6905.
Bansal, A., Singer, J.H., Hwang, B.J., Xu, W., Beaudet, A., and Feller, M.B. (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. Journal of Neuroscience 20, 7672-7681.
Barkis, W.B., Ford, K.J., and Feller, M.B. (2010). Non–cell-autonomous factor induces the transition from excitatory to inhibitory GABA signaling in retina independent of activity. Proceedings of the National Academy of Sciences 107, 22302-22307.
Barros, V.N., Mundim, M., Galindo, L.T., Bittencourt, S., Porcionatto, M., and Mello, L.E. (2015). The pattern of c-Fos expression and its refractory period in the brain of rats and monkeys. Frontiers in cellular neuroscience 9, 72.
Berson, D.M., Dunn, F.A., and Takao, M. (2002). Phototransduction by retinal ganglion cells that set the circadian clock. Science 295, 1070-1073.
Blankenship, A.G., Hamby, A.M., Firl, A., Vyas, S., Maxeiner, S., Willecke, K., and Feller, M.B. (2011). The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina. Journal of Neuroscience 31, 9998-10008.
Bullitt, E. (1990). Expression of c‐fos‐like protein as a marker for neuronal activity following noxious stimulation in the rat. Journal of Comparative Neurology 296, 517-530.
Caval-Holme, F., Zhang, Y., and Feller, M.B. (2019). Gap junction coupling shapes the encoding of light in the developing retina. Current Biology 29, 4024-4035. e4025.
Chew, K.S., Renna, J.M., McNeill, D.S., Fernandez, D.C., Keenan, W.T., Thomsen, M.B., Ecker, J.L., Loevinsohn, G.S., VanDunk, C., and Vicarel, D.C. (2017). A subset of ipRGCs regulates both maturation of the circadian clock and segregation of retinogeniculate projections in mice. Elife 6, e22861.
Clarkson-Townsend, D.A., Bales, K.L., Marsit, C.J., and Pardue, M.T. (2021). Light Environment Influences Developmental Programming of the Metabolic and Visual Systems in Mice. Investigative Ophthalmology & Visual Science 62, 22-22.
Davis, Z.W., Chapman, B., and Cheng, H.-J. (2015). Increasing spontaneous retinal activity before eye opening accelerates the development of geniculate receptive fields. Journal of Neuroscience 35, 14612-14623.
Dkhissi-Benyahya, O., Coutanson, C., Knoblauch, K., Lahouaoui, H., Leviel, V., Rey, C., Bennis, M., and Cooper, H.M. (2013). The absence of melanopsin alters retinal clock function and dopamine regulation by light. Cellular and Molecular Life Sciences 70, 3435-3447.
Drescher, U., Kremoser, C., Handwerker, C., Löschinger, J., Noda, M., and Bonhoeffer, F. (1995). In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359-370.
Feller, M.B., Wellis, D.P., Stellwagen, D., Werblin, F.S., and Shatz, C.J. (1996). Requirement for cholinergic synaptic transmission in the propagation of spontaneous retinal waves. Science 272, 1182-1187.
Ford, K.J., and Feller, M.B. (2012). Assembly and disassembly of a retinal cholinergic network. Visual neuroscience 29, 61-71.
Garaschuk, O., Hanse, E., and Konnerth, A. (1998). Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus. The Journal of physiology 507, 219-236.
Garaschuk, O., Linn, J., Eilers, J., and Konnerth, A. (2000). Large-scale oscillatory calcium waves in the immature cortex. Nature neuroscience 3, 452-459.
Hanson, M.G., and Landmesser, L.T. (2003). Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord. Journal of Neuroscience 23, 587-600.
Hanson, M.G., and Landmesser, L.T. (2004). Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43, 687-701.
Harrison, K.R., Chervenak, A.P., Resnick, S.M., Reifler, A.N., and Wong, K.Y. (2021). Amacrine cells forming gap junctions with intrinsically photosensitive retinal ganglion cells: ipRGC types, neuromodulator contents, and connexin isoform. Investigative Ophthalmology & Visual Science 62, 10-10.
Hattar, S., Liao, H.W., Takao, M., Berson, D.M., and Yau, K.W. (2002). Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295, 1065-1070.
Hattar, S., Lucas, R.J., Mrosovsky, N., Thompson, S., Douglas, R., Hankins, M.W., Lem, J., Biel, M., Hofmann, F., and Foster, R.G. (2003). Melanopsin and rod–cone photoreceptive systems account for all major accessory visual functions in mice. Nature 424, 75-81.
Hornberger, M.R., Dütting, D., Ciossek, T., Yamada, T., Handwerker, C., Lang, S., Weth, F., Huf, J., Weßel, R., and Logan, C. (1999). Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Neuron 22, 731-742.
Johnson, J., Fremeau, R.T., Duncan, J.L., Rentería, R.C., Yang, H., Hua, Z., Liu, X., LaVail, M.M., Edwards, R.H., and Copenhagen, D.R. (2007). Vesicular glutamate transporter 1 is required for photoreceptor synaptic signaling but not for intrinsic visual functions. Journal of Neuroscience 27, 7245-7255.
Kania, A., and Klein, R. (2016). Mechanisms of ephrin–Eph signalling in development, physiology and disease. Nature reviews Molecular cell biology 17, 240-256.
Kinane, C., Calligaro, H., Jandot, A., Coutanson, C., Haddjeri, N., Bennis, M., and Dkhissi-Benyahya, O. (2021). Dopamine desynchronizes the retinal clock through a melanopsin-dependent regulation of acetylcholine retinal waves during development. bioRxiv.
Kirkby, L.A., and Feller, M.B. (2013). Intrinsically photosensitive ganglion cells contribute to plasticity in retinal wave circuits. Proceedings of the National Academy of Sciences 110, 12090-12095.
Lucas, J.A., and Schmidt, T.M. (2019a). Cellular properties of intrinsically photosensitive retinal ganglion cells during postnatal development. Neural Development 14, 1-19.
Lucas, J.A., and Schmidt, T.M. (2019b). Cellular properties of intrinsically photosensitive retinal ganglion cells during postnatal development. Neural Development 14, 8.
McLaughlin, T., Torborg, C.L., Feller, M.B., and O'Leary, D.D. (2003). Retinotopic map refinement requires spontaneous retinal waves during a brief critical period of development. Neuron 40, 1147-1160.
McNeill, D.S., Sheely, C.J., Ecker, J.L., Badea, T.C., Morhardt, D., Guido, W., and Hattar, S. (2011). Development of melanopsin-based irradiance detecting circuitry. Neural development 6, 1-10.
Meister, M., Wong, R.O., Baylor, D.A., and Shatz, C.J. (1991). Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 252, 939-943.
Panda, S., Provencio, I., Tu, D.C., Pires, S.S., Rollag, M.D., Castrucci, A.M., Pletcher, M.T., Sato, T.K., Wiltshire, T., and Andahazy, M. (2003). Melanopsin is required for non-image-forming photic responses in blind mice. Science 301, 525-527.
Petros, T.J., Shrestha, B.R., and Mason, C. (2009). Specificity and sufficiency of EphB1 in driving the ipsilateral retinal projection. Journal of Neuroscience 29, 3463-3474.
Prigge, C.L., Yeh, P.-T., Liou, N.-F., Lee, C.-C., You, S.-F., Liu, L.-L., McNeill, D.S., Chew, K.S., Hattar, S., and Chen, S.-K. (2016). M1 ipRGCs influence visual function through retrograde signaling in the retina. Journal of Neuroscience 36, 7184-7197.
Rao, S., Chun, C., Fan, J., Kofron, J.M., Yang, M.B., Hegde, R.S., Ferrara, N., Copenhagen, D.R., and Lang, R.A. (2013). A direct and melanopsin-dependent fetal light response regulates mouse eye development. Nature 494, 243-246.
Renna, J.M., Chellappa, D.K., Ross, C.L., Stabio, M.E., and Berson, D.M. (2015). Melanopsin ganglion cells extend dendrites into the outer retina during early postnatal development. Developmental neurobiology 75, 935-946.
Renna, J.M., Weng, S., and Berson, D.M. (2011). Light acts through melanopsin to alter retinal waves and segregation of retinogeniculate afferents. Nature neuroscience 14, 827-829.
Sekaran, S., Lupi, D., Jones, S., Sheely, C., Hattar, S., Yau, K.-W., Lucas, R., Foster, R., and Hankins, M. (2005). Melanopsin-dependent photoreception provides earliest light detection in the mammalian retina. Current Biology 15, 1099-1107.
Sherry, D.M., Wang, M.M., Bates, J., and Frishman, L.J. (2003). Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits. Journal of Comparative Neurology 465, 480-498.
Sonoda, T., Lee, S.K., Birnbaumer, L., and Schmidt, T.M. (2018). Melanopsin phototransduction is repurposed by ipRGC subtypes to shape the function of distinct visual circuits. Neuron 99, 754-767. e754.
Sonoda, T., Li, J.Y., Hayes, N.W., Chan, J.C., Okabe, Y., Belin, S., Nawabi, H., and Schmidt, T.M. (2020). A noncanonical inhibitory circuit dampens behavioral sensitivity to light. Science 368, 527-531.
Stellwagen, D., and Shatz, C. (2002). An instructive role for retinal waves in the development of retinogeniculate connectivity. Neuron 33, 357-367.
Sun, C., Warland, D.K., Ballesteros, J.M., Van Der List, D., and Chalupa, L.M. (2008). Retinal waves in mice lacking the β2 subunit of the nicotinic acetylcholine receptor. Proceedings of the National Academy of Sciences 105, 13638-13643.
Syed, M.M., Lee, S., Zheng, J., and Zhou, Z.J. (2004). Stage‐dependent dynamics and modulation of spontaneous waves in the developing rabbit retina. The Journal of physiology 560, 533-549.
Tessier-Lavigne, M., and Goodman, C.S. (1996). The molecular biology of axon guidance. Science 274, 1123-1133.
Tian, N., and Copenhagen, D.R. (2003). Visual stimulation is required for refinement of ON and OFF pathways in postnatal retina. Neuron 39, 85-96.
Triplett, J.W. (2014). Molecular guidance of retinotopic map development in the midbrain. Current Opinion in Neurobiology 24, 7-12.
Tritsch, N.X., Yi, E., Gale, J.E., Glowatzki, E., and Bergles, D.E. (2007). The origin of spontaneous activity in the developing auditory system. Nature 450, 50-55.
Tufford, A.R., Onyak, J.R., Sondereker, K.B., Lucas, J.A., Earley, A.M., Mattar, P., Hattar, S., Schmidt, T.M., Renna, J.M., and Cayouette, M. (2018). Melanopsin retinal ganglion cells regulate cone photoreceptor lamination in the mouse retina. Cell reports 23, 2416-2428.
Wang, L., Rangarajan, K.V., Lawhn-Heath, C.A., Sarnaik, R., Wang, B.-S., Liu, X., and Cang, J. (2009). Direction-specific disruption of subcortical visual behavior and receptive fields in mice lacking the β2 subunit of nicotinic acetylcholine receptor. Journal of Neuroscience 29, 12909-12918.
Watt, A.J., Cuntz, H., Mori, M., Nusser, Z., Sjöström, P.J., and Häusser, M. (2009). Traveling waves in developing cerebellar cortex mediated by asymmetrical Purkinje cell connectivity. Nature neuroscience 12, 463-473.
Wong, R., Chernjavsky, A., Smith, S., and Shatz, C. (1995). Early functional neural networks in the developing retina. Nature 374, 716-718.
Wong, R.O., Meister, M., and Shatz, C.J. (1993). Transient period of correlated bursting activity during development of the mammalian retina. Neuron 11, 923-938.
Yoshida, M., Feng, L., Grimbert, F., Rangarajan, K.V., Buggele, W., Copenhagen, D.R., Cang, J., and Liu, X. (2011). Overexpression of neurotrophin-3 stimulates a second wave of dopaminergic amacrine cell genesis after birth in the mouse retina. Journal of Neuroscience 31, 12663-12673.
Zhang, D.-Q., Belenky, M.A., Sollars, P.J., Pickard, G.E., and McMahon, D.G. (2012). Melanopsin mediates retrograde visual signaling in the retina.
Zhang, D.-Q., Wong, K.Y., Sollars, P.J., Berson, D.M., Pickard, G.E., and McMahon, D.G. (2008). Intraretinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons. Proceedings of the National Academy of Sciences 105, 14181-14186.
Zhang, R.w., Wei, H.p., Xia, Y.m., and Du, J.l. (2010). Development of light response and GABAergic excitation‐to‐inhibition switch in zebrafish retinal ganglion cells. The Journal of physiology 588, 2557-2569.
Zhong, J., Liang, M., Akther, S., Higashida, C., Tsuji, T., and Higashida, H. (2014). c-Fos expression in the paternal mouse brain induced by communicative interaction with maternal mates. Molecular brain 7, 1-11.