內生性光敏感視網膜神經節細胞對感光細胞退化小鼠晚期發育過程中神經活性的影響

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研究生：	陳芃嫻 Chen, Peng-Shian
論文名稱：	內生性光敏感視網膜神經節細胞對感光細胞退化小鼠晚期發育過程中神經活性的影響 The role of intrinsically photosensitive retinal ganglion cells in affecting neural activity during late development of rd1 mice
指導教授：	焦傳金 Chiao, Chuan-Chin
口試委員:	陳令儀 Chen, Lin-yi 陳示國 Chen, Shih-Kuo
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2023
畢業學年度：	111
語文別：	中文
論文頁數：	48
中文關鍵詞：	內在光敏視網膜神經節細胞、藍光刺激、間隙連接耦合、神經再生、視網膜發育
外文關鍵詞：	intrinsically photosensitive retinal ganglion cells, blue light stimulation, gap junction coupling, nerve regeneration, retinal development
相關次數：	點閱：3 下載：0
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在囓齒類動物出生後的早期階段，內在光敏視網膜神經節細胞（ipRGCs）是視桿細胞和視錐細胞成熟前位於神經節細胞層（GCL）的主要感光細胞，可以通過間隙連接將光訊號傳播到其他視網膜神經節細胞，這種早期的光反應可調節晝夜週期並影響睜眼前視網膜神經訊息投射到特定腦區的分布。然而，神經節細胞之間顯著的間隙連接耦合在成年階段消失了，從ipRGCs主導的光反應轉換為視桿細胞/視錐細胞的光反應之時間及機制仍然未知。在這項研究中，多電極陣列 (MEA) 用於記錄野生型 (wt) 和感光細胞退化小鼠 (rd1) 的光誘導放電模式，同時，組織培養也被用來觀察光誘導的神經再生。結果顯示，wt小鼠視網膜的光反應起源於出生後p15~p16的視桿細胞和視錐細胞，出乎意料的是，雖然rd1小鼠在同一階段失去了大部分光感受器，但它們仍然具有對藍光刺激的光感測能力。基於藥理學實驗的結果，我們發現 rd1 小鼠中這種顯著的光反應是由 ipRGCs-RGCs 間隙連接網絡引起的，而在wt視網膜中不完全依賴這種關係。因此，我們的研究不僅支持視桿細胞/視錐細胞的成熟減少了出生後到成年階段神經節細胞之間的間隙連接，並且在透過間隙連接偶聯與否和調節ipRGCs的視網膜發育功能中發揮重要作用。

In the early postnatal stage of rodents, intrinsically photosensitive retinal ganglion cells (ipRGCs), which are the main photosensitive cells before the maturation of rods and cones, can spread the light signal to other retinal ganglion cells via gap junctions located at the ganglion cell layer (GCL). This early photoresponse modulates the day-night cycle and influences the segregation of retinogeniculate projections before eye-opening. However, significant gap junction coupling among ganglion cells disappears at the adult stage. The timing and mechanism of the switch from ipRGCs-dominated light responses to rods/cones-dominated light responses are still unknown. In this study, the multi-electrode array (MEA) was used to record the light-induced firing patterns of wild-type (wt) and retinal degeneration mice (rd1). At the same time, tissue culture was also used to observe light-induced nerve regeneration. The results showed that the light response in the wt mouse’s retina originated from the rods and cones at postnatal p15~p16. Unexpectedly, although the rd1 mice lost most of their photoreceptors at the same stage, they still had the capability of photodetection upon blue light stimulation. Based on the result from the pharmacological experiment, we found that this prominent light response in rd1 mice was caused by the ipRGC-RGC gap junction network, while wt mice didn’t fully rely on this relationship. Thus, our study not only supports that rods/cones maturation reduces the gap junction connection among ganglion cells at the postnatal-to-adult stage, but it also plays an important role in regulating the function of ipRGCs in retinal development through gap junction coupling and decoupling.

摘要----------------------------------------------------------------------------------------------- 2
Abstract------------------------------------------------------------------------------------------ 3
致謝----------------------------------------------------------------------------------------------- 4
Contents------------------------------------------------------------------------------------------ 6
Chapter 1. Introduction----------------------------------------------------------------------- 8
1.1    Retinal degeneration progressively make photoreceptors lose function and death---------------------------------------------------------------------------------------8
1.2    The characteristics and functions of third photosensitive neurons in mammal retinas------------------------------------------------------------------------- 9
1.3    Responses of third photoreceptor neurons to light during developmental stages-------------------------------------------------------------------------------------- 9
1.4    Aims of the present study------------------------------------------------------------ 10
Chapter 2. Materials and methods-------------------------------------------------------- 12
2.1     Retinal explant preparation-------------------------------------------------------- 12
2.2     Retinal explant culture-------------------------------------------------------------- 12
2.3     Neurite outgrowth quantification------------------------------------------------- 13
2.4     Light stimulation for culture------------------------------------------------------- 14
2.5     Immunohistochemistry/ Immunofluorescence of frozen section------------ 14
2.6     Electrophysiological recording and analysis------------------------------------ 15
2.7     Pharmacology treatment----------------------------------------------------------- 16
2.8  Colocalization------------------------------------------------------------------------ 17
2.9  Statistics------------------------------------------------------------------------------- 18
Chapter 3. Results---------------------------------------------------------------------------- 19
3.1  Blue light stimulation promotes neurite outgrowth in retinal explants in p15 rd1 mice------------------------------------------------------------------------------------ 19
3.2  Different light-evoked sustained responses of wt and rd1 ganglion cells at p15~p16 and p20~p21 mice ----------------------------------------------------------------- 19
3.3  The neural activity of melanopsin-expressed RGCs evoked by blue light stimulation spreads across the retina via gap junctions from p15~p21------------ 20
3.4  Rd1 can perceive light stimulation on ganglion cells which has no direct relationship between ON/OFF response ------------------------------------------------- 22
3.5  On rd1 mice, the light response of ipRGC can last longer than wt-------- 23
3.6  Cell number and distribution of melanopsin in wild type (wt) and retinal degeneration mice (rd1) at two different stages----------------------------------------- 24
3.7  Gap junction-mediated coupling with ipRGCs in the inner plexiform layers of the retina---------------------------------------------------------------------------- 24
Chapter 4. Discussion------------------------------------------------------------------------ 26
4.1  Activating ipRGCs to promote neurite outgrowth of postnatal retinal explants in p15 rd1 under blue light stimulation---------------------------------------- 26
4.2  Rd1 ganglion cells were sensitive to blue light at p15~p21------------------ 27
4.3  Gap junctions primarily contribute to the spread of spike responses from ipRGCs to adjacent ipRGCs and other RGCs in rd1 mice--------------------------- 28
Chapter 5. References------------------------------------------------------------------------ 30
Chapter 6. Figures---------------------------------------------------------------------------- 34
                                

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