视网膜感光细胞退变过程中谷氨酸信号通道的变化及其对视网膜内层神经元功能的影响

The function of inner retinal neurons (mainly include bipolar cells and retinal ganglion cells) changed a lot during retinal photoreceptors degeneration. Applying a certain glutamate receptor antagonist inhibited the abnormal spontaneous spiking in retinal ganglion cells and in another case, increased the light sensitivity of ganglion cells, indicating that blocking the glutamate signal pathway may prolong the functional decay of ganglion cells during photoreceptors degeneration. But whether the same effect would be found in bipolar cells, and whether the effect arises from the differential changes of various glutamate receptors are not clear. In this project, I hypothesize that different glutamate signal pathways undergo different changes during photoreceptors degeneration, which will affect the function (includes light responses and responses to glutamate receptor agonist) of inner retinal neurons. And by blocking different glutamate signal pathways by specific antagonist, we may delay the functional decay of inner retinal neurons, thus prolong the time window to treat retinal degeneration. In order to test this hypothesis, we will use rd10 mouse as the animal model for photoreceptors degeneration. At the early, middle and late stages of the degeneration, we will first test the expression and distribution of various glutamate receptors and signal molecules in the inner retinal neurons by immunochemical staining. Then by electrophysiological recording, we will test the functional changes in bipolar cells and retinal ganglion cells to both light stimuli and the puff of glutamate receptor agonist. Next we will apply the antagonist of specific glutamate receptors to see how it changes the response of inner retinal neurons. By this project, we hope to seek a effective strategy to prolong the time window of treating photoreceptors degeneration.

视网膜感光细胞退变时，内层神经元（主要包括双极细胞和节细胞）功能发生变化，而施加特定谷氨酸受体拮抗剂可以抑制节细胞的超正常自发或增强其光反应，提示阻断谷氨酸信号通路可以延缓节细胞的功能退变。但阻断谷氨酸信号通道能否影响双极细胞功能、这些影响是否来自谷氨酸各类受体的不同变化并不清楚。因此，本项目假设感光细胞退变时，谷氨酸各类受体在内层神经元的分布和表达发生了不同变化，从而影响内层神经元的对光反应和谷氨酸兴奋性反应；而通过谷氨酸受体拮抗剂阻断不同的谷氨酸信号通道，可以延缓内层神经元的功能退变。我们将采用视网膜色素变性小鼠模型rd10, 在感光细胞退变的早中晚各时期，通过免疫组化方法检测内层神经元中谷氨酸各受体的分布及表达变化；通过电生理方法检测双极细胞和节细胞的功能变化；进一步施加各谷氨酸通道拮抗剂，检测其对内层神经元的功能影响。此研究对于探索如何延长治疗感光细胞退变的时间窗口具有重要意义。

视网膜色素变性是严重致盲性疾病。本课题旨在检测感光细胞退变过程中视网膜中谷氨酸受体的表达变化及其对视网膜内层神经元功能的影响，以探索治疗视网膜色素变性疾病的新方法。我们使用视网膜色素变性的转基因模型小鼠rd10，通过免疫荧光检测了视网膜退变过程中调控ON信号通路的代谢型谷氨酸受体mGluR6（及其下游）和调控OFF信号通路的离子型谷氨酸受体AMPA、Kainate表达的变化；通过离体多电极阵列记录系统检测视网膜节细胞的光反应变化，以及灌流低浓度的非NMDA离子型谷氨酸受体拮抗剂CNQX的影响；然后通过每日腹腔注射CNQX或GYKI52466（AMPA受体特异性阻断剂）来治疗rd10小鼠，观察治疗后rd10小鼠节细胞的光反应变化、视网膜形态的变化以及动物视觉行为的变化。结果发现感光细胞退变过程中，mGluR6和TRPM1受体表达下降，APMA受体在视网膜外网层分布异常，而AMPA/KA受体在内网层表达上调。节细胞的光反应随着感光细胞退变逐渐降低，且自发异常增高；体外灌流和体内注射CQNX能够降低rd10小鼠节细胞的异常自发、增加节细胞的光反应。体内实验进一步发现CNQX能够增加感光细胞的存活率，改善双极细胞的形态，并提高rd10小鼠的视觉行为。因此，我们的研究表明抑制非NMDA离子型谷氨酸受体能够延缓rd10小鼠节细胞的功能退变，提示此方法可以作为一个有效的策略来延长视网膜色素变性治疗的时间窗口。