Gβ5蛋白在感光細胞及雙極細胞的生理作用上皆扮演關鍵性角色，過去在剔除小鼠中Gβ5蛋白的研究中亦曾發現，雙極細胞的樹突形態及其與感光細胞間的突觸形成皆會因為Gβ5缺失而造成顯著影響。而Gβ5蛋白除了在視網膜外叢狀層有表現外，在視網膜內叢狀層及其節細胞層也有表現，但Gβ5蛋白對視網膜節細胞的影響仍不清楚。因此，在本篇研究中，我們利用基因槍技術將帶有PSD95-GFP之質體隨機轉染至Gβ5基因被選擇性剔除的小鼠視網膜節細胞中，使節細胞樹突上的興奮性突觸位置產生綠色螢光訊號，以進一步探討Gβ5蛋白表現缺失對於小鼠視網膜節細胞的樹突形態及興奮性突觸空間分佈情形的影響。在本篇研究中共收集到五種不同形態的視網膜節細胞，實驗結果發現，雖然Gβ5基因剔除小鼠的視網膜節細胞與控制組小鼠相比之下，樹突形態外觀有些許相異之處，但樹突在內叢狀層的分層位置仍不受影響。而興奮性突觸空間分佈的分析顯示，不管哪種類型的視網膜節細胞，其興奮性突觸在樹突結構上的間隔距離皆為1-2 μm，但在Gβ5基因剔除小鼠的G2、G6和G12這三種類型節細胞的興奮性突觸密度皆稍微低於控制組小鼠。根據上述的實驗結果，不同於Gβ5基因剔除對雙極細胞的樹突形態及突觸形成可產生顯著的影響，Gβ5基因剔除對於節細胞的樹突結構發育及興奮性突觸空間分佈並未造成關鍵性的作用。未來我們將會針對Gβ5蛋白在小鼠視網膜節細胞中的生理功能進行深入研究，以釐清Gβ5蛋白在視覺系統中的功能。

關鍵字: Gβ5蛋白、選擇性基因剔除、樹突形態、突觸分佈、視網膜節細胞

Gβ5 interacts with and stabilizes the R7 members of the regulators of G-protein signaling, which is important for G-protein signaling regulation in photoreceptors and ON-bipolar cells. In addition, it has been found that the dendritic morphology and the number of synaptic triads in the outer plexiform layer (OPL) are significantly altered in the Gβ5-/- mice. However, Gβ5 is also expressed in the inner plexiform layer (IPL) and the ganglion cell layer (GCL). In the present study, we examined the potential role of Gβ5 on dendritic morphology and synaptic distribution of retinal ganglion cells (RGCs). Gβ5 was conditionally knockout (cKO) in the Grik4-cre mice, which selectively label a subset of RGCs. The particle-mediated gene transfer technique was used to transfect RGCs of Gβ5floxed/floxed and Gβ5floxed/+ mice with PSD95-GFP plasmids. The dendritic morphology and the spatial distribution pattern of excitatory synapses of the cKO Gβ5 RGCs were visualized and compared with their littermate controls. Total of 5 RGC types (G2, G6, G12, G17, and G18) were analyzed in the present study. Their dendritic stratifications were normal in the absence of Gβ5, though the dendritic patterns of some RGCs were slightly altered. The PSD95 puncta were regularly spaced (in 1-2 μm interval) on local dendritic segments in both cKO Gβ5 RGCs and their littermate controls. However, the density of excitatory synapses along the dendrites was slightly reduced in G2, G6, and G12 cells of cKO Gβ5 mice compared with their littermate controls. Overall, our results showed that the morphological features of RGCs are only slightly changed when their Gβ5 is conditionally deleted. These findings suggest that, unlike the role of Gβ5 in the OPL (i.e., dramatically affecting the morphology and synapses of ON-bipolar cells), Gβ5 is apparently not essential for the development of dendritic morphology and synaptic distribution in RGCs. The functional significance of Gβ5 expression in the IPL and GCL thus remains to be studied in the future.

Keywords: Gβ5, conditional knockout, dendritic morphology, synaptic distribution, retinal ganglion cell

摘要 i
ABSTRACT iii
CHAPTER 1. INTRODUCTION 1
1-1 Neural circuitry in the retina 1
1-2 Dendritic morphologies and excitatory synaptic patterns in RGCs 2
1-3 The role of Gβ5 in vision 3
1-4 Goals and approaches 5
CHAPTER 2. MATERIALS AND METHODS 6
2-1 Animals 6
2-2 Retina preparation 7
2-3 Preparation of gene gun bullets 8
2-4 Gene gun transfection 9
2-5 Retina culture 9
2-6 Retina fixation 10
2-7 Image acquisition 10
2-8 Classification of RGCs 11
2-9 Image analysis 11
2-10 Statistical analysis 12
CHAPTER 3. RESULTS 13
3-1 RGC dendrites and excitatory synapses visualized by the expression of PSD95-GFP in the Grik4-cre driven tdTomato-expressing mice 13
3-2 Spatial distribution of excitatory synapses along the dendrites of G17 and G18 cells is similar in control and conditional Gβ5 knockout mice 14
3-3 The linear densities of PSD95-GFP puncta of G2, G6, G12 cells in conditional Gβ5 knockout mice are slightly different from those in control 16
3-4 The average PSD95-GFP puncta densities of all cells have no significant difference between conditional Gβ5 knockout mice and control animals 18
CHAPTER 4. DISCUSSION 19
4-1 Gene gun transfection of PSD95-GFP plasmids to identify excitatory synapses of RGCs in the Grik4-cre conditional Gβ5 knockout mice is a low-yield process 19
4-2 The dendritic morphology of RGCs does not show apparent defects in the conditional Gβ5 knockout retinas 21
4-3 The spatial distribution of excitatory synapses is slightly altered in the conditional Gβ5 knockout RGCs 22
4-4 Conclusion 25
References 26
TABLES 29
FIGURES 32
SUPPLEMENTARY DATA 44

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