POWRE: Role of Calcium-Binding Proteins in Synaptic Transmission Between Retinal Amacrine Cells

这项POWRE研究增强奖将使格里森博士有机会探索使用反义技术来研究她的视网膜神经元研究系统的可行性。细胞内钙离子浓度的控制对于钙依赖事件的调节至关重要，例如神经元之间的突触传递。钙稳态在视网膜中尤其重要，因为突触信号相对较慢，因此钙升高可能很大。本研究项目探讨了缓冲钙结合蛋白在神经元钙稳态和突触传递中的作用。格里森博士将使用反义寡核苷酸来阻断主要的缓冲钙结合蛋白Calretinin的表达，然后研究去除该蛋白对突触传递的影响。突触传递将在一种来自鸡视网膜的培养神经元的制备中进行研究，在那里，一种已识别的视网膜神经元类型--无长突细胞--与其他无长突细胞形成突触。分离的突触连接的无长突细胞对可以用两个神经元的同时穿孔膜片电压钳记录进行高分辨率的研究。通过结合分子生物学和电生理学这两项强大的技术，本研究将加深我们对中枢神经系统钙稳态机制的理解。在这个试点项目中开发的反义方法将用于未来的研究，以选择性地移除可能在视网膜突触传递中重要的其他神经元蛋白。

This POWRE Research Enhancement award will give Dr. Gleason the opportunity to explore the feasibility of using antisense techniques to investigate her research system of retinal neurons. Control of calcium concentration in cells is critical for regulation of calcium-dependent events such as synaptic transmission between neurons. Calcium homeostasis is especially important in the retina, where synaptic signaling is relatively slow and therefore calcium elevations can be large. This research project addresses the role that buffering calcium-binding proteins play in neuronal calcium homeostasis and synaptic transmission. Dr. Gleason will use antisense oligonucleotides to block the expression of the predominant buffering calcium-binding protein, calretinin, then examine the effects of removing this protein on synaptic transmission. Synaptic transmission will be studied in a preparation of cultured neurons derived from the chick retina, where an identified type of retinal neuron, the amacrine cell, forms synapses with other amacrine cells. Isolated pairs of synaptically connected amacrine cells can be studied with high resolution using simultaneous perforated-patch voltage-clamp recording of both neurons. By combining the two powerful techniques of molecular biology and electrophysiology, this research will enhance our understanding of calcium-homeostasis mechanisms in the central nervous system. The antisense methods developed in this pilot project will be used in future studies to selectively remove other neuronal proteins that may be important in retinal synaptic transmission.