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

Anderson, G.R., Posokhova, E., and Martemyanov, K.A. (2009). The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling. Cell Biochemistry and Biophysics 54, 33-46.
Badea, T.C., and Nathans, J. (2004). Quantitative analysis of neuronal morphologies in the mouse retina visualized by using a genetically directed reporter. Journal of Comparative Neurology 480, 331-351.
Birnbaumer, L. (2007). Expansion of signal transduction by G proteins. The second 15 years or so: from 3 to 16 alpha subunits plus betagamma dimers. Biochimica et Biophysica Acta 1768, 772-793.
Bleckert, A., Parker, E.D., Kang, Y., Pancaroglu, R., Soto, F., Lewis, R., Craig, A.M., and Wong, R.O. (2013). Spatial relationships between GABAergic and glutamatergic synapses on the dendrites of distinct types of mouse retinal ganglion cells across development. PloS One 8, e69612.
Chen, C.K., Burns, M.E., He, W., Wensel, T.G., Baylor, D.A., and Simon, M.I. (2000). Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature 403, 557-560.
Chen, C.K., Eversole-Cire, P., Zhang, H., Mancino, V., Chen, Y.J., He, W., Wensel, T.G., and Simon, M.I. (2003). Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5. Proceedings of the National Academy of Sciences of the United States of America 100, 6604-6609.
Chen, Y.P., and Chiao, C.C. (2014). Spatial distribution of excitatory synapses on the dendrites of ganglion cells in the mouse retina. PloS One 9, e86159.
Coombs, J., van der List, D., Wang, G.Y., and Chalupa, L.M. (2006). Morphological properties of mouse retinal ganglion cells. Neuroscience 140, 123-136.
Haverkamp, S., Grunert, U., and Wassle, H. (2000). The cone pedicle, a complex synapse in the retina. Neuron 27, 85-95.
Hooks, S.B., Waldo, G.L., Corbitt, J., Bodor, E.T., Krumins, A.M., and Harden, T.K. (2003). RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity. Journal of Biological Chemistry 278, 10087-10093.
Howe, A.K. (2004). Regulation of actin-based cell migration by cAMP/PKA. Biochimica et Biophysica Acta 1692, 159-174.
Ishii, M., and Kurachi, Y. (2003). Physiological actions of regulators of G-protein signaling (RGS) proteins. Life Sciences 74, 163-171.
Ivanova, E., Hwang, G.S., and Pan, Z.H. (2010). Characterization of transgenic mouse lines expressing Cre recombinase in the retina. Neuroscience 165, 233-243.
Jakobs, T.C., Koizumi, A., and Masland, R.H. (2008). The spatial distribution of glutamatergic inputs to dendrites of retinal ganglion cells. Journal of Comparative Neurology 510, 221-236.
Keresztes, G., Martemyanov, K.A., Krispel, C.M., Mutai, H., Yoo, P.J., Maison, S.F., Burns, M.E., Arshavsky, V.Y., and Heller, S. (2004). Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse. Journal of Biological Chemistry 279, 1581-1584.
Kessels, M.M., Schwintzer, L., Schlobinski, D., and Qualmann, B. (2011). Controlling actin cytoskeletal organization and dynamics during neuronal morphogenesis. European Journal of Cell Biology 90, 926-933.
Koizumi, A., Jakobs, T.C., and Masland, R.H. (2011). Regular mosaic of synaptic contacts among three retinal neurons. Journal of Comparative Neurology 519, 341-357.
Koizumi, A., Zeck, G., Ben, Y., Masland, R.H., and Jakobs, T.C. (2007). Organotypic culture of physiologically functional adult mammalian retinas. PloS One 2, e221.
Kong, J.H., Fish, D.R., Rockhill, R.L., and Masland, R.H. (2005). Diversity of ganglion cells in the mouse retina: unsupervised morphological classification and its limits. Journal of Comparative Neurology 489, 293-310.
Krispel, C.M., Chen, C.K., Simon, M.I., and Burns, M.E. (2003). Prolonged photoresponses and defective adaptation in rods of Gbeta5-/- mice. Journal of Neuroscience 23, 6965-6971.
Makino, E.R., Handy, J.W., Li, T., and Arshavsky, V.Y. (1999). The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit. Proceedings of the National Academy of Sciences of the United States of America 96, 1947-1952.
Morgan, J.L., Schubert, T., and Wong, R.O. (2008). Developmental patterning of glutamatergic synapses onto retinal ganglion cells. Neural Development 3, 0-18.
Morgan, J.L., Soto, F., Wong, R.O., and Kerschensteiner, D. (2011). Development of cell type-specific connectivity patterns of converging excitatory axons in the retina. Neuron 71, 1014-1021.
Moritoh, S., Komatsu, Y., Yamamori, T., and Koizumi, A. (2013). Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina. PloS One 8, e54667.
Moritoh, S., Tanaka, K.F., Jouhou, H., Ikenaka, K., and Koizumi, A. (2010). Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. PloS One 5, e12917.
Murphy, D.D., and Segal, M. (1997). Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein. Proceedings of the National Academy of Sciences of the United States of America 94, 1482-1487.
Nakazawa, K., Quirk, M.C., Chitwood, R.A., Watanabe, M., Yeckel, M.F., Sun, L.D., Kato, A., Carr, C.A., Johnston, D., Wilson, M.A., et al. (2002). Requirement for hippocampal CA3 NMDA receptors in associative memory recall. Science 297, 211-218.
Nomura, A., Shigemoto, R., Nakamura, Y., Okamoto, N., Mizuno, N., and Nakanishi, S. (1994). Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell 77, 361-369.
Rao, A., Dallman, R., Henderson, S., and Chen, C.K. (2007). Gbeta5 is required for normal light responses and morphology of retinal ON-bipolar cells. Journal of Neuroscience 27, 14199-14204.
Sun, W., Li, N., and He, S. (2002). Large-scale morphological survey of mouse retinal ganglion cells. Journal of Comparative Neurology 451, 115-126.
Volgyi, B., Chheda, S., and Bloomfield, S.A. (2009). Tracer coupling patterns of the ganglion cell subtypes in the mouse retina. Journal of Comparative Neurology 512, 664-687.
Wassle, H. (2004). Parallel processing in the mammalian retina. Nature Reviews Neuroscience 5, 747-757.
Watson, A.J., Katz, A., and Simon, M.I. (1994). A fifth member of the mammalian G-protein beta-subunit family. Journal of Biological Chemistry 269, 22150-22156.
Wong, W.T., Faulkner-Jones, B.E., Sanes, J.R., and Wong, R.O. (2000). Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho. Journal of Neuroscience 20, 5024-5036.
Wong, W.T., and Wong, R.O. (2000). Rapid dendritic movements during synapse formation and rearrangement. Current Opinion in Neurobiology 10, 118-124.
Yamada, R.X., Matsuki, N., and Ikegaya, Y. (2005). cAMP differentially regulates axonal and dendritic development of dentate granule cells. Journal of Biological Chemistry 280, 38020-38028.