成熟後的哺乳類中樞神經系統在受到嚴重創傷後往往無法再生。然而目前已知在視網膜節細胞的發育過程中，軸突再生能力的逐漸喪失並不是由其本身內在的基因決定，而是受到外在環境的訊息影響。自發性視網膜波為視網膜發育過程中最主要的神經活動，因此藉由模仿發育過程中的神經活性來重現成長時的環境，將有可能對視網膜節細胞的軸突再生有所助益。結果顯示，當我們提供短時間相似於第二期視網膜波的電刺激時，可以有效提升原先應該表現第三期視網膜波的視網膜外植體的神經纖維生長。類似的情形，在同期的視網膜中，藉由短時間阻擋抑制性的神經傳遞進而引發視網膜產生大規模波動狀的神經活動，即使在沒有外加神經營養因子的情況下，視網膜外植體的神經纖維生長亦可明顯增加。更令人意外的結果是，藉由藥物抑制麩胺酸而非乙烯膽鹼的神經傳遞，竟可顯著地減少原先應該表現由乙烯膽鹼主導的第二期視網膜波的視網膜外植體的神經纖維生長。這些發現支持了短期回復視網膜在發育當中前一期的神經活性可有效促進視網膜神經纖維的生長，這些成果也有機會在未來對因節細胞發育過程中逐漸喪失軸突生長能力的視網膜提供有效的治療方法。

關鍵字 ： 視網膜波、視網膜節細胞、電刺激、軸突再生、神經營養因子

Mature mammalian central nervous system fails to regenerate after severe injuries. However, it is known that gradual loss of axon growth ability of retinal ganglion cells (RGCs) during development is largely determined by extrinsic signals rather than programmed genetically. Spontaneous retinal waves are the major neural activity during retinal development. Thus, restoring developmental environment by mimicking these neural activities may provide help to axon regeneration of RGCs. Here we demonstrated that neurite outgrowth of retinal explants was greatly enhanced when short term electrical stimulation similar to stage II retinal waves was applied to the retinas normally displayed stage III retinal waves. Similarly, short term induction of globally wave-like neural activity by blocking inhibitory neural transmission in the same age of retinas also greatly promoted neurite outgrowth even in the absence of exogenous neurotrophic factors. Surprisingly, neurite outgrowth was significantly reduced when glutamate transmission was blocked pharmacologically in the retinas normally displayed stage II cholinergic retinal waves. These findings support that short term restoration of environments that mimic neural activities of developmentally precedent retinas is sufficient to enhance neurite outgrowth of retinal explants, and may lead to a therapeutic strategy of preventing the gradual loss of axon growth ability of RGCs in more mature retinas.
Key words: retinal waves, retinal ganglion cells, electrical stimulation, axon regeneration, neurotrophic factors

Contents
摘要 i
Abstract ii
1. Introduction 1
2. Materials and Methods 5
2.1 Retinal explant preparation 5
2.2 Retinal explant culture 5
2.3 Immunohistochemistry 6
2.4 Neurite outgrowth identification and quantification 7
2.5 Electrical stimulation and recording 8
2.6 Pharmacological treatments 9
2.7 Cell viability assay 9
2.8 Wave property analysis 10
3. Results 12
3.1 Stage II wave-like electrical stimultaion promotes survival and neurite outgrowth in retinal explants 12
3.2 Short term electrical stimulation without altering intrinsic wave properties is sufficient to promote neurite outgrowth in P11 retinal explants 13
3.3 Glutamate transmission but not stage II retinal waves promotes neurite outgrowth in P5 retinal explants 14
3.4 Short term induction of globally wave-like neural activity promotes survival and neurite outgrowth in P11 retinal explants 16
4. Discussion 18
4.1 Glutamatergic transmission affects neurite outgrowth in immature retinal explants 18
4.2 ES promotes neurite outgrowth by stimulating RGCs directly 20
4.3 Short-term elevation of neural activity results in long-term effect of promoting neurite outgrowth 21
4.4 The promoting effect of neurite outgrowth depends on electrical activity patterns 22
4.5 Limitation of neurite outgrowth promoted by neural activity 24
5. References 25
6. Figures 29

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