| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 107 === During a critical period of visual circuit refinement, stage II retinal waves are initiated by the release from starburst amacrine cells (SACs), propagating throughout the layer containing retinal ganglion cells (RGCs). We previously found that RGCs may release glutamate diffusely in the inner plexiform layer (IPL) and ganglion cell layer (GCL), thus modulating stage II retinal waves and further regulating the eye-specific segregation of the dorsal lateral geniculate nucleus (dLGN) in thalamus. However, the detail in glutamate regulation of stage II retinal waves remains unclear. First, to investigate how glutamate transmission affects retinal waves in vivo, we performed the intraocular injection of iGluR antagonists in rat pups. We found that wave frequency and spike time tiling coefficient (STTC) across distance were downregulated by the intraocular blockade of iGluR in vivo, suggesting that glutamate transmission modulates wave properties in vivo. Additionally, we found that the expression level of AMPA subunit 2 (GluA2) was increased upon the intraocular application of iGluR antagonists in vivo, suggesting that the inhibition of glutamate transmission may lead to the alterations in gene expression during stage II retinal waves. Second, to determine the presence of the glutamate receptor in developing retinas, we performed immunofluorescence staining and found that the GluA2 was mainly expressed in the IPL and GCL. Further, to investigate the role of glutamate autocrine regulation via RGCs, we transfected the anti-sense GLUA2 to specifically knockdown the GluA2 expression in RGCs. We found that wave frequency and STTC across distance were upregulated by GluA2 depletion, suggesting that glutamate receptors may mediate the autocrine regulation via RGCs, thus regulating the wave frequency and firing correlation. Third, to examine whether the GluA2 dynamics on plasma membrane can be changed upon different stimuli, we transfected the developing retinas with the optical reporter of GluA2 trafficking (i.e., the fusion protein of pHluorin and GluA2), we found that GluA2 internalization was promoted by application of CGS 21680, a selective agonist of adenosine A2A receptor shown to increase wave frequency via SACs. Furthermore, we found that GluA2 externalization was promoted by the minute level of glutamate or by iGluR antagonists. Our results suggest that wave activity may regulate the dynamics of GluA2 trafficking, and the homeostasis of glutamate transmission is maintained by a critical level of transmitter-receptor interaction during stage II retinal waves. Together, our data suggest that glutamate transmission during the stage II wave period may act via RGCs, through AMPA receptors. The dynamics of AMPA receptors in RGCs may play an important role in modulating stage II retinal waves during retinal circuit refinement.
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